Translational Pharmacokinetic and Pharmacodynamic Evaluation of Mefloquine Against Plasmodium falciparum Using a Hollow Fiber Infection Model

Abstract

This study established a plastic-based hollow fiber infection model (HFIM) to reproduce human-like mefloquine exposure and quantify antimalarial pharmacodynamics (PD) against blood-stage Plasmodium falciparum. Asynchronized ring-stage 3D7 parasites were maintained in the extracapillary space under continuous-flow culture conditions that supported sustained growth across two consecutive 48 h intraerythrocytic life cycles. The observed parasitemia dynamics were comparable in static and dynamic drug-free controls. Target total plasma concentration time profiles for intravenous mefloquine were generated using a pharmacokinetic (PK) simulation study based on previously reported compartmental models from adult malaria patients and subsequently implemented in the HFIM through programmed dilution to mimic first-order elimination, with dosing simulated as a 100 mg intravenous (IV) bolus every 24 h for 3 days. The HFIM exposure profile maintained clinically relevant peak concentrations with the long clinical PK half-life of mefloquine. Under these dynamic exposure conditions, mefloquine produced sustained suppression of parasitemia across successive life cycles relative to both control conditions, consistent with concentration-driven blood-stage activity. The estimated IC50 was 0.03 mg/L. Together, this study demonstrated that the commonly used plastic-based HFIM can simulate clinically relevant PK profiles and characterize the antimalarial PD against blood-stage P. falciparum. This addresses limitations of static in vitro assays and reduces the reliance on animal models, where preclinical-to-clinical concordance can be poor. Thereby, HFIM provides a promising framework for optimizing antimalarial dosing strategies. Future studies will extend this approach to rational evaluation of combination therapies using PK/PD-guided regimen design to improve clinical relevance.

Over the past decade, substantial progress has been made toward reducing the global burden of malaria. Nevertheless, Plasmodium falciparum malaria remains a major global health challenge. According to the most recent malaria report, an estimated 282 million malaria cases and approximately 610,000 deaths were reported in 2024, representing an increase compared with 2023. Children under five years of age continue to bear the greatest burden, accounting for nearly 76% of malaria-related deaths, particularly in high-transmission regions.

Chemoprophylaxis and preventive chemotherapy play critical roles in reducing malaria transmission and associated morbidity and mortality. However, the emergence and spread of P. falciparum resistance increasingly complicate both treatment and prevention strategies. To mitigate resistance and improve clinical outcomes, the World Health Organization recommends artemisinin-based combination therapies (ACTs) as first-line treatment for uncomplicated P. falciparum malaria. Optimization of existing antimalarial combinations and exploration of alternative dosing strategies rely heavily on preclinical in vitro systems that complement clinical investigation.

Conventional in vitro antimalarial assays typically use static drug concentrations and therefore do not reproduce the rising and falling concentrations of human pharmacokinetic (PK) profiles, thereby limiting their translational relevance. These limitations are particularly important for antimalarial agents whose efficacy is driven by dynamic, time-varying exposure rather than constant concentrations.

To address this gap, we developed a plastic-based hollow fiber infection model (HFIM) capable of simulating human-like drug exposure and quantitatively evaluating antimalarial pharmacodynamics in vitro. The HFIM allows precise control of concentration–time profiles, providing a robust and translational platform for characterizing pharmacokinetic–pharmacodynamic (PK/PD) relationships under clinically relevant conditions. , Prior studies used a highly labor-intensive glass-based HFIM model to study the PK/PD of antimalarial drugs. , The glass-based HFIM developed by Shapiro et al. demonstrated the ability to simulate clinically relevant drug exposure profiles over extended experimental durations (typically 96–120 h), enabling characterization of parasite responses across multiple intraerythrocytic life cycles. Building on these approaches, the present plastic-based HFIM offers distinct practical advantages, , including improved scalability, reduced complexity, and enhanced experimental reproducibility, while maintaining the ability to capture clinically relevant PK/PD dynamics. Given that most antimalarial drugs act against blood-stage parasites residing within human red blood cells, parasitemia reduction is a biologically and clinically meaningful end point that can be directly captured in this dynamic system.

P. falciparum, the most virulent human malaria parasite, readily lends itself to in vitro cultivation and serves as a robust model organism for antimalarial drug evaluation. , Accordingly, this study was designed to address two primary objectives: (i) to establish and maintain the intraerythrocytic life cycle of P. falciparum within a plastic-based hollow fiber cartridge over two complete 48 h replication cycles, and (ii) to characterize the pharmacodynamic effects of mefloquine on parasite development across approximately 1.5 asexual life cycles. This experimental approach enables precise simulation of clinically relevant drug exposure profiles and provides critical insight into stage-specific antimalarial activity.

Among the many unusual features of the malaria parasite, P. falciparum exhibits a tightly regulated 48 h asexual intraerythrocytic developmental cycle, culminating in the synchronous rupture of infected red blood cells and release of merozoites. − This periodicity is governed by an intrinsic parasite oscillator and is associated with molecular signatures linked to circadian and intraerythrocytic cycle–specific regulatory programs. During this asexual cycle, parasites progress sequentially through the ring, trophozoite, and schizont stages. Early ring-stage parasites predominate in peripheral circulation and are therefore the most readily observed manifestation of the 48 h life cycle in human infections.

Although early ring-stage P. falciparum parasites are less metabolically active and intrinsically less susceptible to antimalarial agents compared with trophozoites and schizonts, they represent the primary circulating form in vivo and are consequently the first stage exposed to therapeutic drug concentrations. − Importantly, drug effects initiated during the ring stage frequently manifest later as impaired trophozoite maturation, disrupted schizogony, or failure of parasite replication. Accordingly, evaluation of ring-stage parasites provides a clinically relevant and mechanistically informative framework for characterizing antimalarial pharmacodynamics and predicting in vivo efficacy. ,

In erythrocytic malaria parasites, a notable consequence of targeting essential metabolic pathways, particularly those associated with the apicoplast, is delayed death. Parasites with compromised apicoplast function are often able to complete the first 48 h asexual cycle; however, they fail to sustain viability in the subsequent cycle, ultimately leading to parasite clearance. As a result, antimalarial efficacy becomes more pronounced when drug exposure is extended across multiple 48 h life cycles, highlighting the importance of prolonged and dynamic exposure conditions in pharmacodynamic evaluation. ,

Materials and Methods

Parasite Cultivation

Asexual blood-stage P. falciparum strain 3D7 (MRA-1001) was maintained in continuous in vitro culture using human AB⁺ erythrocytes at a hematocrit of 2%–4%. Human AB⁺ erythrocytes were obtained from LifeSouth Community Blood Centers. All blood products were screened and confirmed to be negative or nonreactive for all FDA-required infectious disease markers (e.g., HIV, hepatitis B virus, and hepatitis C virus), using FDA-licensed testing assays prior to use. Parasites were cultured in RPMI 1640 medium supplemented with 25 mM HEPES, 2 g/L sodium bicarbonate, 0.5% (w/v) Albumax II, 0.1 mM hypoxanthine, and 20 U/mL penicillin–streptomycin. Cultures were incubated at 37 °C under a controlled gas atmosphere consisting of 5% CO₂, 5% O₂, and 90% N₂.

Parasitemia was routinely monitored by microscopic examination of Giemsa-stained thin blood smears, and cultures were maintained at parasitemia levels below 10% to prevent nutrient depletion. Fresh erythrocytes and complete medium were replenished as needed to maintain parasite viability and exponential growth. Parasite cultures were maintained under standard laboratory conditions without synchronization, resulting in an asynchronous population comprising mixed developmental stages, and were used as the starting inoculum for all subsequent experiments.

In Vitro Static Drug Concentration Experiments

In vitro 6-well plate experiments with static drug concentrations were conducted in parallel with the dynamic drug-concentration hollow fiber infection model studies. This allowed us to compare the no-drug control and treated arms between both systems and to establish baseline parasite growth under constant culture conditions. Asynchronized ring-stage P. falciparum parasites were cultured in complete RPMI using human AB⁺ erythrocytes under standard incubation conditions with a regulated gas mix. Daily broth medium changes were necessary to maintain adequate oxygenation. Parasitemia was monitored over time by Giemsa-stained thin blood smears and expressed as the percentage of infected erythrocytes.

Parasitemia Setup for Hollow Fiber Infection Model

For HFIM experiments with dynamic simulated drug concentrations, asynchronized ring-stage P. falciparum parasites were adjusted to a starting parasitemia of 2.1%–2.5% at a hematocrit of 2.4% prior to inoculation into the extracapillary space of the hollow fiber cartridge. , All HFIM studies were conducted using C7025 cartridges (Batch no.720060222, FiberCell Systems, Inc.). These conditions were selected to establish a stable baseline parasitemia at the start of each experiment. Following inoculation, parasite cultures were maintained under continuous-flow conditions to ensure adequate nutrient exchange, oxygen supply, and efficient removal of metabolic waste, thereby supporting sustained parasite viability and replication throughout multiple 48 h intraerythrocytic life cycles. , Parasite viability and growth were monitored throughout the experiment by serial sampling from the extracapillary space of the HFIM cartridge. Parasitemia was quantified by microscopic examination of Giemsa-stained thin blood smears and expressed as the percentage of infected erythrocytes relative to the total number of erythrocytes counted, with a minimum of 1000 cells evaluated per slide.

General Overview of HFIM

The plasma concentration–time profiles of mefloquine following intravenous bolus administration of 100 mg every 24 h were simulated in the HFIM at 37 °C over 3 days. Experiments were conducted in duplicate on separate occasions to assess reproducibility. This study was conducted as a proof-of-concept to assess the feasibility of using the plastic-based HFIM to investigate antimalarial PK and PD in P. falciparum (Figure ).

1.

Schematic representation of the hollow fiber infection model (HFIM) used to simulate human pharmacokinetics and evaluate pharmacodynamic responses against Plasmodium falciparum. (1) Target drug concentration–time profiles corresponding to clinically relevant doses are first identified using literature data or pharmacokinetic simulations and implemented through the dosing input. (2) The central reservoir volume (V) is defined, and the elimination flow rate is calculated (Pump flow = Ke × V) to reproduce the desired drug half-life; parallel pumps may be incorporated to simulate active metabolites or combination therapies. (3) The hollow fiber cartridge is inoculated with P. falciparum at approximately 2.5% parasitemia and 2.4% hematocrit and maintained under continuous gas flow (∼5% CO₂, ∼5% O₂, balance N₂) at 37 °C to support optimal parasite growth. (4) Drug-containing medium circulates through the system to mimic dynamic in vivo exposure, while serial pharmacokinetic (PK) and pharmacodynamic (PD) samples are collected over time to quantify drug concentrations and parasite response.

Simulating Target PK Profiles in the HFIM

As a proof-of-concept chemotherapeutic agent for uncomplicated P. falciparum malaria, mefloquine was selected based on its long half-life and well-characterized human PK. Target human exposure profiles for intravenous mefloquine were simulated using PumasAI (Pumas, Julia) based on a one-compartment PK model, based on published population PK parametrized with previously reported data from adult malaria patients, including a clearance of 1.42 L/h and a volume of distribution of 625 L. Mefloquine was dosed every 24 h as an IV bolus input.

Intravenous dosing in the HFIM was implemented as an instantaneous bolus delivered into the central reservoir to achieve the desired initial concentration. A mefloquine 1 mg/mL stock solution was prepared in 100% DMSO. Immediately prior to dosing, the stock solution was diluted in complete RPMI to the target concentration. The final DMSO concentration in broth was maintained below 0.1% (v/v), a level considered noncytotoxic.

The mefloquine bolus was introduced into the primary circulation loop connecting the central reservoir containing 200 mL of complete RPMI to the intracapillary space of the hollow fiber cartridge. The circulation flow rate was set to 20 mL/min to ensure rapid equilibration of mefloquine between the central reservoir and the cartridge.

Experiments were conducted using a small hydrophilic polyvinylidene fluoride hollow fiber cartridge with a nominal pore size of 0.03 μm (C7025, FiberCell Systems). Mefloquine recovery was evaluated by comparing concentrations measured from the hollow fiber cartridge with dynamically generated control concentrations calculated from the target infusion profile over the experimental period. Differences between groups were analyzed using a two-sample t-test, with statistical significance defined as p < 0.05. As shown in Supplementary Figure S1, no significant differences were observed between the measured cartridge concentrations and the calculated control concentrations across both low and high mefloquine concentrations, indicating minimal nonspecific binding of mefloquine to the hollow fiber cartridge and supporting reproduction of the intended concentration–time profile.

Target Pharmacokinetic Exposure

Throughout the experimental design, target mefloquine exposure was defined using the free (unbound) drug concentrations reported in the literature. However, mefloquine exhibits extensive and nonlinear binding because of its high affinity for erythrocyte membrane lipids (reported dissociation constant for erythrocyte membrane lipid binding, K_D ≈ 3 × 10̂(−7)­M, equivalent to 0.1135 mg/L). Parasites were maintained in RPMI 1640 medium supplemented with 0.8% Albumax II, a lipid-rich bovine serum albumin preparation that provides a biomimetic protein–lipid binding environment and partially reproduces the lipophilic binding capacity of human plasma. Although Albumax II does not fully replicate human plasma composition, its lipid-associated binding properties provide a physiologically relevant matrix for highly lipophilic compounds such as mefloquine. To account for drug binding within this experimental system, the free fraction of mefloquine was estimated using the nonlinear protein-binding model described by ter Heine et al.

$$\mathrm{F}\text{\_}\mathrm{u}=\mathrm{C}\text{\_}\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{e}/\mathrm{C}\text{\_}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}=(\mathrm{K}\text{\_}\mathrm{D}+\mathrm{C}\text{\_}\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{e})/(\mathrm{B}\text{\_}\max +\mathrm{K}\text{\_}\mathrm{D}+\mathrm{C}\text{\_}\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{e})$$ 1

where F_uis the free drug fraction, C_freeis the free drug concentration, C_totalis the total drug concentration, B_maxis the maximum binding capacity, and K_Dis the dissociation constant. The estimated free mefloquine concentrations were subsequently used to establish the target pharmacokinetic concentration–time profile in the HFIM.

Plasma Drug and Parasitemia Sampling

Mefloquine concentrations in the HFIM were determined at 0, 0.5, 1, 4, 8, 12, 18, 24, 32, 48, 56, and 72 h following dosing to characterize the concentration–time profile and reproduce the target concentration profiles in patients (i.e., plasma). Samples were collected from the central reservoir and stored under appropriate conditions until analysis. In pilot studies, additional samples were collected from both the cartridge and the central reservoir to evaluate potential discrepancies in drug concentrations between the compartments. In parallel, PD sampling was performed to quantify parasite response. Parasitemia percentages were determined at 0, 4, 8, 18, 24, 32, 48, 56, 68, 72, 78, 90, and 96 h for both control and mefloquine-treated conditions, enabling assessment of parasite growth and drug effects across multiple 48 h intraerythrocytic life cycles.

Bioanalysis

Calibration standard curves were prepared by spiking 20 mL of a series of working standard solutions into 180 mL of complete RPMI to achieve the desired concentrations. For Mefloquine samples and standards, 200 μL of each sample or standard was mixed with 800 μL of methanol containing diclofenac (200 ng/mL, internal standard). The mixtures were then centrifuged at 13,000 rpm for 10 min to precipitate the proteins. Following centrifugation, the supernatant was injected into the UPLC-MS/MS system.

The concentrations were determined using an Acquity I-Class UPLC system (Waters, Milford, MA) interfaced with a Triple Quad 6500+ MS/MS system (AB Sciex, Framingham, MA). The UPLC column used was an Acquity UPLC BEH C18 (130 Å, 2.1 × 100 mm, 1.7 μm; Waters, Milford, MA, USA). For Mefloquine, separation was carried out at 45 °C with a run time of 3.5 min. The mobile phase consisted of 2 mM ammonium formate in water (A) and acetonitrile (B), at a flow rate of 0.4 mL/min. The gradient was as follows: 5% B (0 to 1.0 min), 5%–70% B (1.0–1.2 min), 70%–95% B (1.2–2.4 min), 95%–5% B (2.4–2.5 min), 5% B (2.5–3.5 min). The injection volume was 10 μL. The MS/MS system was operated in both positive-ion modes using the TurboSpray IonDriveTM. Source/gas parameters were: curtain gas, 30 psi; collision gas, 9 psi; ion spray voltage, +5500 V; source temperature, 550 °C; ion source gas 1 and 2, both at 35 psi. The optimized multiple reaction monitoring (MRM) transitions were m/z 379.0→321.2 for Mefloquine, and m/z 296.0→215.1 for diclofenac. Dwell time was 20 ms for all compounds.

Peak integration and data analysis were performed in Analyst software (AB Sciex, Framingham, MA). Samples were quantified against calibration curves generated in each analytical batch. Assay performance was validated based on established acceptance criteria. Assay precision/accuracy (mean %) were as follows: 4.0%/0.4% at 2 μmol/L mefloquine, 6.6%/2.4% at 0.5 μmol/L, 9.2%/2.8% at 0.01 μmol/L, and 4.3%/2.0% at 0.002 μmol/L (lower limit of quantification [LLOQ]).

Mathematical Modeling of Drug Concentrations and Parasitemia in the HFIM

Pharmacokinetic-Pharmacodynamic (PKPD) analyses were conducted with MonolixSuite version 2024R1 using the mixed-effects models method. A two-compartmental model with linear elimination described the drug concentration–time profile. These concentrations in the HFIM were compared to the clinical PK data.

These in vitro PK parameters were further integrated into a PKPD model to predict efficacy. The indirect turnover model described the drug effect with full inhibition (Imax = 1), where R0 is the baseline effect (i.e., in the absence of drug), IC50 indicates the half-maximal inhibitory concentration, and Kout is the degradation rate constant, describing the natural loss of parasitemia over time, which is fixed at 0.06 h^–1 (eq ). The Stochastic approximation method is used to estimate all PKPD parameters.

$$dR/dt=Ki{n}^{*}(1-\frac{[MQ]}{[MQ]+IC50})-Kou{t}^{*}R$$ 2

Results

Using a published population pharmacokinetic model developed from patients with uncomplicated P. falciparum malaria, simulated peak plasma concentrations were approximately 1.2 mg/L following a single oral 500 mg dose and approximately 0.25 mg/L following a single oral 100 mg dose (Figure ).

2.

Simulated plasma concentration–time profiles of intravenous mefloquine following single bolus doses of 100 mg (left panel) and 500 mg (right panel). Concentration–time profiles were generated using a pharmacokinetic simulation study based on previously reported parameters from adult malaria patients and illustrate the rapid distribution phase followed by the prolonged terminal elimination characteristic of mefloquine.

These simulated concentration–time profiles were subsequently used as the target exposures for the HFIM experiments. The PK of mefloquine in the HFIM was linear with an estimated clearance of 0.95 L/h, and the volume of distribution was 460 L (Table ).

1. Mefloquine PK/PD Parameters in an in vitro System (HFIM).

parameter	unit	literature value	estimate	relative SE (%)	95% confidence interval
total clearance, CL	L/h	1.49 2.77	0.95	7.46	0.82–1.1
volume of central compartment, V1	L	476 359	459.92	1.46	446.97–473.25
volume of peripheral compartment, V2	L	860 474	290.31	13.4	224.2–375.92
intercompartmental clearance, Q	L/h	85.7 11.7	22.07	17.9	15.65–31.11
baseline response, R 0	%		17.23	9.41	14.34–20.69
half-maximal inhibitory concentration, IC50	mg/L		0.0123	37.7	0.0077–0.0168
	nM		32.5		20.5–44.5

Simulated drug concentrations in the HFIM for 100 mg mefloquine dosed every 24 h showed that the HFIM could adequately capture the mefloquine peak concentrations and long half-life in patients (Figure ). There was no significant difference in drug concentration between samples collected from the cartridge and the central reservoir in our experiment, and this is also supported by the literature.

3.

Observed mefloquine concentration–time profile in the hollow fiber infection model, with the black line representing the mean and the blue shaded region indicating ±standard deviation (SD).

In the absence of the drug, parasitemia growth in the dynamic control medium was comparable to that observed in the static control medium throughout the experiment (Figure ). In both control conditions, parasitemia followed the characteristic 48 h intraerythrocytic developmental cycle of P. falciparum, with peak parasitemia observed between 8 and 18 h within each cycle. Parasitemia growth in the dynamic control medium was comparable to that observed under static conditions, with both systems exhibiting peak parasitemia of approximately 12%–14% during the first cycle and maintaining a similar temporal pattern during the second cycle, reaching comparable levels at corresponding time points (Figure ).

4.

Parasitemia percentage of Plasmodium falciparum 3D7 under static and dynamic control conditions and following mefloquine (100 mg) exposure.

Mefloquine exposure markedly suppressed parasite growth, limiting peak parasitemia to ∼6% to 7% during the first 48 h cycle and to ≤4% during the second cycle. At 72 h, parasitemia in the mefloquine-treated system was reduced by 55%–60% relative to both control conditions. The extensive PD effect of mefloquine supports the assumption in the PK/PD model that it can fully inhibit parasite replication (Imax = 100%). The estimated growth rate constant of P. falciparum in vitro was 0.72 day^–1, and mefloquine was estimated to have an IC50 of 0.0193 mg/L or 32.5 nM (Table ). These results demonstrate sustained mefloquine efficacy across consecutive life cycles under clinically relevant exposure conditions in the hollow fiber infection model. Across all conditions, ring-stage parasites predominated in the circulating population, consistent with expected blood-stage dynamics under drug-free conditions.

Discussion

Mefloquine is a quinoline–methanol antimalarial whose precise mechanism of action has long remained unclear. Recent evidence, however, suggests that mefloquine binds directly to the cytoplasmic 80S ribosome (Pf80S) of P. falciparum, thereby inhibiting protein synthesis and exerting potent blood-stage schizonticidal activity. , This mechanism is consistent with its broad activity against asexual blood-stage parasites and supports its classification as a concentration-driven antimalarial agent. For these reasons, together with its well-characterized human pharmacokinetic profile and chemical stability under in vitro culture conditions, mefloquine was selected as the proof-of-concept compound for establishing and validating the HFIM. Although mefloquine monotherapy is no longer routinely recommended due to concerns about resistance and dose-related neurological adverse effects, it remains a key partner drug in the World Health Organization (WHO)-recommended artesunate–mefloquine combination therapy for uncomplicated P. falciparum malaria. Therefore, validation of the HFIM using mefloquine provides a clinically relevant foundation for future studies aimed at optimizing artesunate–mefloquine and other antimalarial combination regimens. Pharmacokinetically, mefloquine is characterized by a larger volume of distribution and a long terminal half-life of about 2–3 weeks in humans. ,, These properties contribute to marked interspecies variability observed in preclinical models, complicating translational interpretation from animal studies and underscoring the need for alternative translational platforms.

In this study, the HFIM successfully reproduced a human-like PK profile of mefloquine, capturing both peak exposure and the long half-life consistent with clinical observations. , The HFIM’s ability to maintain sustained exposure over multiple 48 h intraerythrocytic cycles represents a significant advantage for evaluating drugs such as mefloquine, whose efficacy depends on prolonged exposure rather than rapid parasite clearance alone. This proof-of-concept demonstrates that the plastic-based HFIM established here can overcome limitations associated with conventional static in vitro assays and interspecies discrepancies inherent to animal models. Compared with the previously described glass-based HFIM, the plastic cartridge system is less costly, easier to assemble, and enables repeated serial sampling from a single experimental unit, thereby reducing experimental complexity and increasing the practicality of HFIM studies for antimalarial PK/PD investigations.

Under drug-free conditions, P. falciparum exhibited exponential growth following inoculation, with parasitemia following the characteristic 48 h asexual life cycle. The in vitro growth rate of P. falciparum was approximately 0.72 day^–1, consistent with reported in vivo estimates, and experiments were conducted over 96 h to capture two complete intraerythrocytic life cycles. Although two complete intraerythrocytic life cycles of the P. falciparum 3D7 strain were successfully reproduced, the anticipated fold increase in parasitemia during the second cycle was not observed, consistent with prior in vitro observations reported by Nkhoma et al. Peak parasitemia was consistently observed approximately 8–18 h into each cycle, corresponding to ring-stage predominance, followed by a decline as parasites progressed through later developmental stages. , These growth dynamics were preserved across successive cycles, confirming the biological fidelity and stability of the HFIM under optimized culture conditions. Importantly, similar parasite growth patterns were observed for 3D7 under static and dynamic control conditions, indicating that continuous flow and medium exchange alone did not adversely affect parasite viability. ,

Mefloquine exposure resulted in significant suppression of parasite growth across consecutive life cycles. Although modest increases in parasitemia were observed early following inoculation, peak parasitemia in the mefloquine-treated system remained substantially lower than in control conditions and did not recover during the second 48 h cycle. This sustained suppression is consistent with mefloquine’s long half-life and concentration-dependent blood-stage activity and supports the hypothesis that early exposure during the ring stage leads to downstream impairment of parasite maturation and replication. The observed PD response aligns with a turnover model, which could well describe the parasite growth and antimalarial efficacy of mefloquine in the present study. An IC₅₀ of 32.5 nM indicates potent inhibition of P. falciparum growth by mefloquine and is consistent with previously reported in vitro values. This value aligns with estimated peak concentrations observed in the HFIM and in clinical settings, supporting a direct PK–PD relationship and the translational validity of the HFIM-derived pharmacodynamic response. −

Collectively, these findings demonstrate that the plastic-based HFIM can simultaneously capture clinically relevant PK and PD of mefloquine in a single integrated system. Compared with murine malaria models, where parasite clearance kinetics often correlate poorly with human outcomes, the HFIM offers a translationally beneficial platform for evaluating blood-stage antimalarial efficacy. The ability to reproduce both exposure and response under controlled conditions represents a major advantage for rational dose selection and regimen optimization.

Despite these strengths, several limitations should be acknowledged. The present study successfully simulated the PK of a single-agent over 72 h. However, future studies need to demonstrate the feasibility of simulating longer study durations and the PK of antimalarial drugs with different half-lives. In addition, the current HFIM does not incorporate a hepatic metabolic component. Nevertheless, because mefloquine is pharmacologically active as the parent compound and its metabolites contribute minimally to antimalarial activity, reproducing the clinically observed parent drug concentration–time profile is appropriate for evaluating blood-stage pharmacodynamics in this proof-of-concept study. Future development of the platform may incorporate additional physiological processes in which metabolite exposure is expected to influence the drug’s efficacy or toxicity.

Future studies will focus on leveraging this platform to evaluate rational antimalarial combination therapies, building on our prior success in mimicking oral exposure profiles within the HFIM. Integration of translational PKPD modeling with dynamic in vitro experimentation will enable systematic optimization of dosing strategies that maximize antimalarial killing while minimizing resistance selection. Ultimately, this approach has the potential to substantially improve the development of safe, effective, and clinically relevant antimalarial regimens.

Conclusion

In conclusion, the employed plastic-based HFIM provides a robust and efficient in vitro platform for sustaining P. falciparum growth and evaluating antimalarial drug activity under dynamic PK exposure profiles. The system reliably supports parasite replication across successive 48 h intraerythrocytic life cycles and enables direct observation of drug-induced effects on blood-stage parasitemia. By accurately reproducing clinically relevant PK profiles, the HFIM allows integration of PK and PD. This dynamic in vitro model offers substantial advantages over conventional static assays and animal models, including improved control of drug exposure and reduced reliance on in vivo studies. Thus, the plastic-based HFIM supports the optimization of antimalarial treatment strategies and drug development against P. falciparum.

Glossary

Abbreviations

HFIM

hollow fiber infection model

IV

intravenous

LC-MS/MS

liquid chromatography–tandem mass spectrometry

MQ

mefloquine

PD

pharmacodynamics

PK

pharmacokinetics

PK/PD

pharmacokinetic/pharmacodynamic

CL

total clearance

V1

volume of the central compartment

V2

volume of the peripheral compartment

Q

intercompartmental clearance

R0

baseline response

IC50

half-maximal inhibitory concentration.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.6c00482.

• Figure S1, recovery of mefloquine from the plastic hollow fiber cartridge compared with dynamically generated control concentrations (PDF)

F.J.A.: Conceptualization, Methodology, Supervision, Project administration, Funding acquisition, Resources, Formal analysis, Writingreview and editing. S.R.M.: Methodology, Investigation, Formal analysis, Data curation, Visualization, Writingoriginal draft. K.P.: Investigation, Methodology. L.L.: Methodology, Investigation, Formal analysis, Writingreview and editing. Y.Z.: Methodology, Investigation, Formal analysis, Writingreview and editing. Y.L.: Methodology, Investigation, Formal analysis, Writingreview and editing. J.B.B.: Resources, Formal analysis, Supervision, Writingreview and editing.

The authors declare no competing financial interest.