Abstract
ELQ-300 is a preclinical candidate that targets the liver and blood stages of Plasmodium falciparum, as well as the forms that are crucial to transmission of disease: gametocytes, zygotes, and ookinetes. A significant obstacle to the clinical development of ELQ-300 is related to its physicochemical properties. Its relatively poor aqueous solubility and high crystallinity limit absorption to the degree that only low blood concentrations can be achieved following oral dosing. While these low blood concentrations are sufficient for therapy, the levels are too low to establish an acceptable safety margin required by regulatory agencies for clinical development. One way to address the challenging physicochemical properties of ELQ-300 is through the development of prodrugs. Here, we profile ELQ-337, a bioreversible O-linked carbonate ester prodrug of the parent molecule. At the molar equivalent dose of 3 mg/kg of body weight, the delivery of ELQ-300 from ELQ-337 is enhanced by 3- to 4-fold, reaching a maximum concentration of drug in serum (Cmax) of 5.9 μM by 6 h after oral administration, and unlike ELQ-300 at any dose, ELQ-337 provides single-dose cures of patent malaria infections in mice at low-single-digit milligram per kilogram doses. Our findings show that the prodrug strategy represents a viable approach to overcome the physicochemical limitations of ELQ-300 to deliver the active drug to the bloodstream at concentrations sufficient for safety and toxicology studies, as well as achieving single-dose cures.
INTRODUCTION
Malaria is a potentially fatal tropical disease that strikes ∼200 million people each year, and in 2013, it is estimated to have caused 584,000 deaths (1). The human toll of malaria suffering occurs mostly in sub-Saharan Africa (90% of worldwide cases), where >80% of the victims are children under 5 years of age or expectant mothers. While the metrics for malaria infection and death have been steadily improving over the past decade due to increased use of bed nets and combination medications, there is growing concern from Southeast Asia owing to reports of increasing tolerance exhibited by malaria parasites for the artemisinin class of drugs, resulting in extended clinical response times and treatment failures (2, 3). Because health care providers have turned to artemisinin combination therapies to counter the worldwide spread of chloroquine resistance, there is a pressing need to develop safe and affordable drugs for treatment and prevention of malaria (4). ELQ-300 (Fig. 1) is a preclinical drug candidate that targets the liver and blood stages of Plasmodium falciparum and also kills the sexual and vector stage parasites (i.e., gametocytes, zygotes, ookinetes, and oocysts) that are crucial to disease transmission (5, 6). In murine models of malaria, a single oral dose of 0.03 mg/kg of body weight prevented sporozoite-induced infections, while 4 daily doses of 1 mg/kg achieved complete cures of patent infections. In a feeding study, it was shown that oocyst formation was completely inhibited in mosquitoes that fed on animals dosed with as little as 0.1 mg/kg of ELQ-300.
FIG 1.
Chemical structures of the compounds used or referred to in this study. Atovaquone targets the Plasmodium cytochrome bc1 complex at the Qo site. It is coformulated with proguanil for clinical use to enhance performance and prevent resistance. ELQ-300 is a preclinical candidate and an investigational antimalarial drug that also selectively inhibits the parasite's cytochrome bc1 complex but instead targets the Qi site.
ELQ-300 is a subnanomolar inhibitor of the cytochrome bc1 complex of Plasmodium parasites. The effects of the drug are parasiticidal due to inhibition of the coenzyme Q cycle that is required for de novo pyrimidine biosynthesis (7). The clinical success of atovaquone (i.e., the atovaquone-proguanil formulation known as Malarone) has validated cytochrome bc1 as a target for therapeutic exploitation (Fig. 1) (8). Unfortunately, the high cost of atovaquone manufacture, coupled with a high propensity for the acquisition of resistance in malaria parasites, serves as a barrier to the use of atovaquone-proguanil in widespread treatment campaigns. The risk of emergence of ELQ-300 resistance in Plasmodium parasites appears to be low, since the propensity for resistance in vitro is far less than that observed for atovaquone. Also, in clinical use (e.g., treatment, chemoprophylaxis, or single-dose cures), ELQ-300 would be delivered in combination with other antimalarials to improve efficacy and performance and to delay or prevent resistance in the field. The primary risk to the successful development of ELQ-300 for use in humans involves its physicochemical properties. Oral absorption is limited by relatively poor water solubility and high crystallinity (5). Prior studies showed that oral absorption of ELQ-300 at low doses in the therapeutic range, e.g., 0.1 to 1 mg/kg formulated in undiluted polyethylene glycol 400 (PEG 400), is good. Unfortunately, its poor aqueous solubility has so far limited the oral absorption and blood exposure at higher doses needed to achieve single-dose cures and to establish an acceptable therapeutic window. We believe that the strong tendency of ELQ-300 to form crystals with high lattice strength (i.e., a melting point of >300°C) contributes to its precipitation in gastric fluids when administered in a cosolvent formulation, such as PEG 400, which in turn leads to diminished absorption as the dose is increased above the therapeutic level. In light of its challenging physiochemical properties, a prodrug approach was undertaken to address the high crystallinity of the preclinical candidate in order to enhance oral bioavailability, increase blood exposure, and achieve single-dose cures.
MATERIALS AND METHODS
Chemical synthesis of ELQ-337.
To a flame-dried 50-ml round-bottom flask, 0.5 g ELQ-300 (1.05 mmol; 1 eq), 84 mg sodium hydride (60% dispersion; 2.1 mmol; 2 eq), and 5 ml anhydrous tetrahydrofuran were added. The resulting suspension was heated and stirred at 60°C under an argon atmosphere for 30 min or until a clear solution was obtained. The reaction mixture was removed from the heat, and 200 μl ethyl chloroformate (2.1 mmol; 2 eq) was added dropwise via syringe, resulting in an immediate precipitation of white solids. The suspension was stirred for 5 min and then quenched by dropwise addition of water. The reaction mixture was diluted with water (5 ml) and extracted with ethyl acetate (5 ml 3 times). The organic layer was washed with brine (5 ml) and dried over MgSO4. The residue after evaporation was recrystallized (dichloromethane; hexanes) to give 0.558 g ELQ-337 (97%) as white microcrystals.
X-ray crystallography of ELQ-337.
Diffraction intensities for ELQ-337 were collected at 100 K on a Bruker Apex charge-coupled-device (CCD) diffractometer using MoKα radiation (λ = 0.71073 Å). Space groups were determined and assigned based on systematic absences. Absorption corrections were made using SADABS (Siemens area detector absorption correction software) (9). Structures were solved by direct methods and Fourier techniques and refined on F2 using full matrix least-squares procedures. All non-H atoms were refined with anisotropic thermal parameters. All H atoms were found from the residual density map and refined with isotropic thermal parameters. All calculations were performed with the Bruker SHELXTL (v. 6.10) package (10).
In vitro metabolic stability assays.
Test agents (controls and ELQ-337) were incubated in duplicate with pooled microsomes at 37°C. Each reaction mixture contained microsomal protein in 100 mM potassium phosphate, 2 mM NADPH, 3 mM MgCl2 at pH 7.4. Controls were included for each test agent omitting NADPH to detect NADPH-free degradation. At the indicated times, an aliquot was removed from each experimental and control reaction mixture and mixed with an equal volume of ice-cold stop solution (methanol containing an internal standard). The stopped reaction mixtures were incubated for 10 min at −20°C, and an additional volume of water was added. The samples were centrifuged to remove precipitated protein, and the supernatants were analyzed by liquid chromatography-tandem mass spectrometry (LC–MS-MS) to quantitate the remaining parent compound. The data were converted to the percent remaining by dividing by the time zero concentration value. The appearance of the parent of the prodrug was also measured and compared to a one-point standard. The data were fitted to a first-order decay model to determine the half-life (T1/2). Intrinsic clearance (CLint) was calculated from the half-life and the protein concentrations as follows: CLint = ln2/(T1/2 [microsomal protein]).
Isolation of P. falciparum mitochondria and ubiquinol-cytochrome c oxidoreductase assay.
Parasite mitochondria were isolated by differential centrifugation according to the protocol of Mather et al. (11), which involves a magnetic separation step to remove residual hemozoin. The ubiquinol-cytochrome c reductase activity of the cytochrome bc1 complex in mitochondrial preparations was assayed spectrophotometrically at 35°C in a stirred cuvette, as described previously (5, 11).
In vivo pharmacokinetics in mice.
All animal studies were conducted using established procedures in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and the study protocols were reviewed and approved by the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee.
The systemic exposure of ELQ-300 following oral administration of ELQ-300 and ELQ-337 was studied in nonfasted female Swiss outbred mice (21.6 to 28.8 g) with access to food and water ad libitum at all times. Solution formulations were prepared by suspending solid compound in PEG 400 and sonicating for approximately 60 min. Solutions were administered orally by gavage (0.1 ml per mouse) within 1 h of preparation. Blood samples were collected up to 72 h postdose (n = 3 mice per time point), with a maximum of two samples obtained from each mouse, via either submandibular bleed or terminal cardiac puncture under anesthesia. Blood was collected directly into heparinized polypropylene tubes containing a cocktail of Complete (a protease inhibitor cocktail; Roche Diagnostics), potassium fluoride, and EDTA to minimize the potential for ex vivo degradation of compounds in blood/plasma samples. Once collected, blood samples were centrifuged immediately, and the supernatant plasma was removed. The plasma samples collected following dosing of ELQ-337 were dispensed into polypropylene Eppendorf tubes containing 1 M acetic acid (5 μl per tube) to minimize the potential for ex vivo conversion to ELQ-300. All plasma samples were snap-frozen on dry ice and subsequently stored at −80°C until analysis within 6 weeks. Following protein precipitation with acetonitrile (2-fold volume ratio), plasma samples were analyzed via ultraperformance liquid chromatography (UPLC)-MS (Waters Micromass Quattro Premier coupled to an Acquity UPLC device operating in positive electrospray ionization multiple-reaction monitoring mode). Analyte concentrations were determined relative to ELQ-300 and ELQ-337 calibration curves prepared in blank mouse plasma.
Parasite culture and drug sensitivity.
Laboratory strains of P. falciparum were cultured in human erythrocytes by standard methods under a low-oxygen atmosphere (5% O2, 5% CO2, 90% N2) in an environmental chamber (12). The culture medium was RPMI 1640, supplemented with 25 mM HEPES buffer, 25 mg/liter gentamicin sulfate, 45 mg/liter hypoxanthine, 10 mM glucose, 2 mM glutamine, and 0.5% Albumax II (Life Technologies, Grand Island, NY) (complete medium). The parasites were maintained in fresh human erythrocytes suspended at a 2% hematocrit in complete medium at 37°C. Stock cultures were subpassaged every 3 or 4 days by transfer of infected red cells to a flask containing complete medium and uninfected erythrocytes. The SYBR green I fluorescence-based method was employed to compare the antiplasmodial activity of ELQ-337 to that of ELQ-300 (13). Experiments were set up in triplicate in 96-well plates (Costar; Corning) with 2-fold dilutions of each drug across the plate in a total volume of 100 μl and at a final red blood cell concentration of 2% (vol/vol). The dilution series ranged from 0.25 nM to 250 nM. Automated pipetting and dilution were carried out with the aid of a programmable Precision 2000 robotic station (BioTek, Winooski, VT). An initial parasitemia of 0.2% was attained by addition of normal uninfected red blood cells to a stock culture of asynchronous parasite-infected red blood cells (PRBC). The plates were incubated for 72 h at 37°C in an atmosphere of 5% O2, 5% CO2, 90% N2. After this period, the SYBR green I dye-detergent mixture (100 μl) was added, and the plates were incubated at room temperature for 1 h in the dark and then placed in a 96-well fluorescence plate reader (Spectramax Gemini-EM; Molecular Diagnostics) for analysis, with excitation and emission wavelength bands centered at 497 and 520 nm, respectively. The fluorescence readings were plotted against the logarithm of the drug concentration, and curve fitting by nonlinear regression analysis (GraphPad Prism software) yielded the drug concentration that produced 50% of the observed decline relative to the maximum readings in drug-free control wells (50% inhibitory concentration [IC50]).
In vivo efficacy against murine malaria (Peters 4-day test).
The in vivo activity of ELQ-337 was assessed against the blood stages using a modified 4-day test (14). Mice (female; CF1; Charles River Laboratories) were infected intravenously with 2.5 × 104 to 5.0 × 104 Plasmodium yoelii (Kenya strain; MR4 MRA-428) parasitized erythrocytes from a donor animal. Drug administration commenced the day after the animals were inoculated (day 1). The test compounds were dissolved in PEG 400 and administered by oral gavage once daily for 4 successive days; chloroquine was used as a positive control. On the 5th day, blood films were prepared, and the extent of parasitemia was determined by microscopic examination of Giemsa-stained smears. The 50% effective dose (ED50) and ED90 values (mg/kg/day) were derived graphically from the dose required to reduce the parasite burden by 50% and 90%, respectively, relative to drug-free controls. Animals remaining parasite free 30 days after the last drug dose were considered cured of their infections. The malaria infection in this model system is rapidly fulminate, producing average parasitemias of 30% in untreated control animals by day 5. The procedures involved, together with all matters relating to the care, handling, and housing of the animals used in this study, were approved by the Portland VA Medical Center Institutional Animal Care and Use Committee.
In vivo single-dose efficacy against murine malaria (single-dose cure).
The in vivo activity of ELQ-337 was assessed against the blood stages. Mice (female; CF1; Charles River Laboratories) were infected intravenously with 2.5 × 104 to 5.0 × 104 P. yoelii (Kenya strain; MR4 MRA-428) parasitized erythrocytes from a donor animal. Drug administration commenced the day after the animals were inoculated (day 1). The test compounds were dissolved in PEG 400 and administered by oral gavage once. On the 5th day, blood films were prepared, and the extent of parasitemia was determined by microscopic examination of Giemsa-stained smears. Animals remaining parasite free 30 days after the last drug dose were considered cured of their infection.
RESULTS
Rationale for an ELQ-300 prodrug.
Earlier attempts to develop a prodrug of ELQ-300 were unsuccessful, as they were either too unstable in physiological media (e.g., the acetyl ester) or too stable to release the drug (e.g., 4-O-linked dimethyl-carbamate). In the approach described here, we prepared a series of carbonate esters of ELQ-300. We were interested in the carbonate ester linkage because of our successful use of this feature to enhance the delivery and efficacy of ELQ-121 (15). The crystal structure of ELQ-121 revealed extensive pi-pi stacking of overlapping aromatic quinolone rings in the Z-plane and an extensive network of intermolecular hydrogen bonds in the X-Y plane between the keto oxygen of one quinolone and the ring N-H of an adjacent system (6). In this paper, we focus on one of these prodrugs, ELQ-337. As shown in Fig. 2, attachment of the carbonate promoiety to the 4-position oxygen removes the hydrogen atom from the ring nitrogen, thereby preventing hydrogen bonding and reducing the crystal lattice strength.
FIG 2.
Regioselective chemical synthesis of ELQ-337, an O-linked carbonate ester of ELQ-300.
Chemical synthesis of ELQ-337, the ethyl carbonate ester of ELQ-300.
ELQ-337 was produced from ELQ-300 in one step, using sodium hydride and ethyl chloroformate in tetrahydrofuran. The conversion was complete within minutes upon addition of the chloroformate, forming one regioisomer in very high yield (Fig. 2). Unlike ELQ-300, which decomposes at ∼314°C, ELQ-337 exhibits a melting point of 160°C, indicative of a significant loss of crystal lattice strength.
Molecular structure of ELQ-337.
As typical spectroscopic techniques proved inadequate for unambiguously assigning the regiochemical structure of the product (i.e., 4-position carbonate versus the corresponding N-linked carbamate), ELQ-337 crystals were submitted for X-ray diffraction analysis. Refinement of the X-ray structure revealed that ELQ-337 is a 4-position carbonate ester. It is notable that crystallography confirms the complete absence of hydrogen bonds in the crystal structure, i.e., the carbonyl-to-ring N-H hydrogen bond has been eliminated (Fig. 3). X-ray crystallography also revealed the nearly perpendicular orientation (82.01° of rotation relative to the planar quinoline ring) of the innermost phenyl ring of the side chain (Fig. 3). Additional details can be found in the supplemental material.
FIG 3.
(Left) ORTEP diagram of ELQ-337. Ellipsoids are drawn at the 30% probability level. (Right) A fragment of the single-crystal lattice structure of ELQ-337. It is noteworthy that the X-ray diffraction analysis of ELQ-337 crystals showed that (i) the promoiety is located at position 4 of the quinoline ring, (ii) the innermost phenyl ring attached to position 3 is rotated nearly perpendicular to the planar quinoline ring, and (iii) there is a lack of intermolecular H bonding between adjacent ELQ-337 molecules in the crystal lattice.
ELQ-337 is a weak inhibitor of P. falciparum cytochrome bc1.
The introduction of an ethyl carbonate moiety to the 4 position of ELQ-300 introduces steric bulk and aromatizes the 4(1H)-quinolone core into a 4-oxoquinoline. We previously demonstrated that ELQ-300 is a potent inhibitor of purified P. falciparum cytochrome bc1 with an IC50 of 0.56 nM (5). Using the same assay conditions, we evaluated ELQ-337 for inhibition of P. falciparum cytochrome bc1 and found that enzyme-inhibitory activity is greatly reduced, i.e., by >10,000-fold (IC50 = 10 μM) (Fig. 4). These results show that ELQ-337 has minimal inhibitory activity as a prodrug and has to be converted to ELQ-300 for its antimalarial activity.
FIG 4.
IC50 curves for ELQ-300 and ELQ-337 against P. falciparum cytochrome bc1. Cytochrome c reductase activity was monitored by spectroscopic analysis with a dual-wavelength spectrophotometer in dual mode (550 to 541 nm). The assay was performed at 35°C in a stirred cuvette with horse heart cytochrome c in a buffered solution. Reactions were initiated by additions of hemozoin-free mitochondrial preparation, inhibitor, and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzohydroquinone (decylubiquinol). ELQ-300 and ELQ-337 concentrations are expressed in nanomolar units.
In vitro metabolism of ELQ-337 to ELQ-300.
Carbonate esters are common promoieties for drugs in clinical use; they are stable to acid and labile to alkaline conditions and are enzymatically cleaved by esterase action. We evaluated the metabolism of ELQ-337 in vitro in the presence of human and murine hepatic microsomes. Briefly, the compound was incubated with pooled microsomes at 37°C in the presence or absence of NADPH to establish if metabolism is P450 mediated. Samples were taken at various times of incubation over the course of an hour and analyzed by LC–MS-MS to quantitate the remaining prodrug, as well as the appearance of the parent drug, ELQ-300. In these studies verapamil and warfarin served as control compounds, with high and low metabolic clearance, respectively. As shown in Table 1, ELQ-337 is rapidly converted to ELQ-300 by the enzymatic activity contained in both human and murine hepatic microsomal extracts. The fact that the conversion occurred even in the absence of NADPH supports the notion that esterase activity is involved.
TABLE 1.
In vitro microsomal intrinsic clearance assays
| Compound | Test concn (μM) | Test species | NADPH dependent |
NADPH free |
Comments | ||
|---|---|---|---|---|---|---|---|
| CLint (μl min−1 mg−1) | T1/2 (min) | CLint (μl min−1 mg−1) | T1/2 (min) | ||||
| ELQ-337 | 1 | Human | 53.8 | 12.9 | 57.8 | 12 | Conversion to ELQ-300 tracked by LC–MS-MS |
| Mouse | 128.8 | 5.4 | 157.9 | 4.4 | |||
| Verapamil | 1 | Human | 201 | 11 | 1 | >60 | Highly metabolized control |
| Mouse | 354 | 7 | 3 | >60 | |||
| Warfarin | 1 | Human | 0 | >60 | 0 | >60 | Low-metabolized control |
| Mouse | 0 | >60 | 0.3 | >60 | |||
In vitro activity of ELQ-337.
In vitro experiments showed that the intrinsic antiplasmodial activity of the ethyl carbonate ester ELQ-337 is virtually indistinguishable from that of ELQ-300, with IC50s against all test strains (P. falciparum D6, Dd2, and Tm90-C2B) in the low nanomolar range (Table 2). Given that the prodrug is a comparatively weak inhibitor of the parasite cytochrome bc1 (Fig. 4), these data suggest that ELQ-337 is converted to ELQ-300 by nonspecific esterase activity within parasite-infected red blood cells, an activity apparently absent in isolated parasite mitochondria.
TABLE 2.
Comparative in vitro activities and in vivo efficacies of ELQ-300, ELQ-337, and chloroquine
| Compound |
In vitro activity vs. P. falciparum (IC50) (nM)a |
In vivo efficacy (oral) vs. P. yoelii (mg/kg/day) |
|||||
|---|---|---|---|---|---|---|---|
| D6 | Dd2 | C2B | ED50b | ED90b | NRDc | Single-dose cure | |
| ELQ-300 | 1.7 | 2.5 | 2.3 | 0.02 | 0.06 | 1.0 | >20d |
| ELQ-337 | 1.4 | 2.8 | 2.9 | 0.02e | 0.05e | 1.0e | 2.0e |
| Chloroquine | 5.2 | 95 | 120 | 1.1 | 1.8 | >64 | >64 |
Seventy-two-hour SYBR green assay.
Modified 4-day Peters test (P. yoelii, Kenya strain, MR4).
NRD, non-recrudescence dose.
Solubility limit of ELQ-300 in PEG 400.
The dose for ELQ-337 reflects the molar equivalent dose relative to ELQ-300.
In vivo efficacy of ELQ-337 against murine malaria.
In vivo experiments using the 4-day suppression test protocol, i.e., inoculation (day 0) followed by oral dosing on 4 sequential days with smears on day 5, against P. yoelii infection in mice showed that the action profile of ELQ-337 is essentially the same as that reported previously for ELQ-300 (ED50 = 0.02 mg/kg/day; ED90 = 0.05 mg/kg/day; ED99 = 0.075 mg/kg/day). In addition, the nonrecrudescence dose for ELQ-337 remained impressive, with 30-day cures recorded in the range of 0.3 to 1 mg/kg/day. We were also interested in evaluating the efficacy of ELQ-337 in delivering single-dose cures. Previously, we found that ELQ-300 was unable to cure infected animals with a single dose in the range of 1 to 20 mg/kg when administered in neat PEG 400 (note that 20 mg/kg is the highest dose achievable as a solution with this compound). Here, we show (Table 2) that, unlike ELQ-300, the ethyl carbonate ester provided 4/4 single-dose cures at oral doses as low as 2.3 mg/kg (molar equivalent to 2 mg/kg of the parent ELQ-300), with animals surviving up to 30 days postinfection. Microscopic examination of Giemsa-stained blood smears revealed that the 1-mg/kg dose failed in all four animals on day 12 of the study, i.e., parasites reappeared in the blood 11 days following the single-dose treatment.
ELQ-300 exposure following oral administration of the carbonate ethyl ester prodrug, ELQ-337.
In an attempt to understand the improved efficacy of the prodrug ELQ-337 over ELQ-300 in vivo for achieving potent single-dose cures of infected animals, a pharmacokinetic investigation was conducted in nonfasted female outbred mice. The compounds were dosed as a single oral dose of 3 mg/kg (ELQ-300) and an equivalent molar dose of ELQ-337 (i.e., 3.5 mg/kg), with each formulated in neat PEG 400. Following oral administration of ELQ-337, there were no measurable concentrations of the intact prodrug in plasma, suggesting either that the prodrug was not absorbed intact from the gastrointestinal lumen or that it underwent extensive presystemic degradation. In contrast, there was significant exposure of ELQ-300 (Table 3 and Fig. 5) following administration of ELQ-337. As shown in Fig. 5, oral administration of ELQ-337 at 3.5 mg/kg achieved an ∼3-fold-higher plasma exposure of ELQ-300 compared to data obtained following oral administration of ELQ-300 itself at an equivalent molar dose. Importantly, the maximum concentration of drug in serum (Cmax) for ELQ-300 following a single oral 3.5-mg/kg dose of ELQ-337 was 5.9 μM, a value that is roughly 1,000-fold higher than the measured antiplasmodial EC50 (5). Thus, it appears that the superior one-dose efficacy provided by ELQ-337 can be explained on the basis of improved oral absorption and enhanced blood exposure compared to direct administration of ELQ-300.
TABLE 3.
Exposure parameters for ELQ-300 in female Swiss outbred mice following oral administration of ELQ-300 or prodrug ELQ-337
| Parametera | Value following oral administration of: |
|
|---|---|---|
| ELQ-300 | ELQ-337 | |
| Dose (mg/kg) | 3.0 | 3.5 |
| Cmax (μM) | 2.0 ± 0.3 | 5.9 ± 0.8 |
| AUC0–72 (μM · h) | 64 ± 6 | 258 ± 10 |
| AUC0–∞ (μM · h) | 67 | 302 |
AUC0–72, area under the concentration-time curve from 0 to 72 h; AUC0–∞, area under the concentration-time curve from 0 h to infinity.
FIG 5.
Plasma ELQ-300 concentrations in female Swiss outbred mice following oral administration of ELQ-300 (solid circles) or the prodrug ELQ-337 (open circles). Both compounds were administered as solutions in PEG 400 at molar doses equivalent to 3.0 mg/kg of ELQ-300. The data points represent individual measurements (n = 3 mice) at each sampling time, with the lines drawn through the means.
DISCUSSION
ELQ-300 is a preclinical antimalarial candidate with potent activity against all life cycle stages of P. falciparum, the causative agent of the deadliest form of human malaria. Despite its excellent antimalarial profile, the clinical development of ELQ-300 is hindered by poor aqueous solubility and high crystallinity, both undesirable features that combine to limit exposure at concentrations sufficient to establish a therapeutic window in preclinical animal species. These challenging physicochemical properties also limit the attainment of blood concentrations necessary to achieve single-dose cures. In this report, we describe the facile chemical conversion of ELQ-300 to a bioreversible ethyl carbonate ester with diminished crystallinity and enhanced relative oral bioavailability. A single oral dose of 2.3 mg/kg of the prodrug ELQ-337 was completely curative in mice harboring a patent infection. Taken together, these results show that the prodrug strategy is a viable approach to enhance the delivery and overall efficacy of 4(1H)-quinolones for treatment and prophylaxis of malaria. Furthermore, it appears that ELQ-337 and similar carbonate esters have the potential to be used for single-exposure cure, prophylaxis, and transmission blocking to support a global assault on falciparum malaria.
It is noteworthy that for the experiments described in this report PEG 400 was used to predissolve ELQ-337 for administration to mice. However, for use in treatment and prevention of malaria in humans, it would be necessary to formulate the drug in a stable tablet form. Various formulation strategies are being investigated to enhance the solubility and dissolution of ELQ-337 in gastrointestinal fluid in order to increase oral bioavailability from the solid state. Such strategies include particle size reduction (e.g., nanomilling), solid dispersions (e.g., spray-dried dispersions), and salt formation (16). These approaches are being taken to facilitate compound progression through preclinical testing to allow the assessment of the therapeutic window of the parent compound, ELQ-300. Given that the drug will be used primarily in poverty-stricken regions of the world where malaria is endemic and health care resources are limited, it is important for the final product to be inexpensive and effective in delivering the drug by oral means in a predictable dose-linear fashion.
Looking ahead, we foresee the possibility of using an ELQ-300 prodrug in combination with at least one other antimalarial drug to prolong clinical utility and to delay the emergence of resistant parasites. Given the predicted long in vivo half-life for ELQ-300 in humans, the choice of a combination partner would be informed by the comparative pharmacokinetic and pharmacodynamic properties of the partner drugs. These parameters would be critical for delivering single-dose cures of malaria, in which an ELQ-300 prodrug could play an important role.
Supplementary Material
ACKNOWLEDGMENTS
This project received financial support from the National Institutes of Health (grants AI079182 and AI100569 to M.K.R. and AI028398 to A.B.V.) and from the U.S. Department of Defense Peer Reviewed Medical Research Program (PR130649 to M.K.R.). M.K.R. also receives funding from the U.S. Department of Veterans Affairs Merit Review Program.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01183-15.
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