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Knowledge of pharmacokinetic/pharmacodynamic (PK/PD) relationships can enhance the speed and economy of drug development by enabling informed and rational decisions at every step, from lead selection to clinical dosing. For anti-infective agents in particular, dynamic in vitro hollow fiber cartridge experiments permit exquisite control of kinetic parameters and the study of their consequent impact on pharmacodynamic efficacy. Such information is of great interest for the cost-restricted but much-needed development of new antimalarial drugs, especially since major human pathogen Plasmodium falciparum can be cultivated in vitro but is not readily available in animal models. This protocol describes the materials and procedures for determining the PK/PD relationships of antimalarial compounds.
Malaria is a leading cause of death in many tropical and subtropical countries, reaching highest mortality in young children in Africa (World Health Organization, 2014). Resistance to existing antimalarials, including the last major line of defense artemisinins, makes the need for new drugs greater than ever. High throughput screenings of massive compound libraries against Plasmodium falciparum have identified thousands of potential leads (Gamo et al., 2010, Guiguemde et al., 2010; Anthony et al., 2012) and there is a need for stringent and rational criteria by which to identify the best candidates for further development, especially given the limited resources available for this indication. The importance of pharmacokinetic/pharmacodynamic (PK/PD) relationships in early GO/NOGO decisions is generally acknowledged (Cartwright et al., 2010), and anti-infective programs in particular rely on dynamic in vitro hollow fiber PK/PD studies (Drusano, 2004; Ambrose et al., 2007). Once human PKs are defined, in vitro models based on actual clinical data have proven valuable in predicting best dosing regimens or best drug combinations (Brown et al., 2011; Drusano et al., 2014). Such data can substantially shortcut time and expense. Unfortunately, the ability to study and exploit PK/PD for antimalarials has been limited by lack of a suitable in vitro system, a problem remedied by the apparatus and methods described here.
Several species of Plasmodium cause disease in humans, but falciparum is most pathogenic and the only one reliably cultivated in vitro. The methods described herein focus on the intraerythrocytic forms of P. falciparum that evolve through a 48 hr cycle, and cause clinically apparent disease (Shapiro and Goldberg, 2006). The described methods are tailored for economical and standardized discovery of the PKs that govern a compound’s antimalarial efficacy. Several aspects of this methodology differ from those used for other anti-infectives. First, and key to the success of this work, was development of a glass cartridge system to replace commercial hollow fiber cartridges that are toxic to P. falciparum (Bakshi et al., 2013). Second, P. falciparum was acclimated to grow in conventional 5% CO2 incubator conditions rather than the more typical 2–5% O2 (Bakshi et al., 2013). Third, since the potency of a compound may vary as the parasite progresses through its 48 hr cycle (Delves et al., 2012), these studies utilize asynchronous cells akin to most falciparum populations in vivo (Fairhurst and Wellems, 2010; Dondorp and von Seilein, 2010), and run for 72 hr to cover 1.5 lifecycles. Finally, while the applied PKs (peak concentration, half-life) can be programmed to simulate those in animals or humans, they need not do so. Indeed, as described below a distinct advantage of the system is that artificial conditions, independent of those imposed by animal physiology, can be applied so as to discern the fundamental PK driver of efficacy.
Orally administered drugs are subject to absorption, distribution, metabolism, and elimination (Rowland and Tozer, 2004). These result in blood levels that rise to a peak then fall with a given half-life (Fig. 1A). Area under the concentration-time curve (AUC) is a measure of total drug exposure. By varying the dosing regimen, the same AUC can be obtained with concentration-time curves of widely differing shapes. At the extremes are a concentration-intensive short-lived high peak versus a time-intensive long-lived constant lower concentration infusion (Fig. 1B). As perhaps best characterized for anti-bacterials, efficacy for a given drug class is preferentially driven by either concentration or time of exposure (Ambrose et al., 2007). This unit describes the methods for discerning the PK governance of antimalarial activity.
Each experiment involves three glass cartridges: two drug-treated and one drug-free control (‘drug’ may denote any compound of interest). Malaria parasites are exposed to two dosing regimens that differ widely in their concentration-time profile, but nevertheless provide the same total exposure (AUC, Fig. 1B). The governing PK parameter (CMAX or TMIC) is identified based on the antimalarial efficacy of a large rapidly cleared peak (CMAX-driven regimen) versus that of a constant infusion (TMIC-driven regimen; see Video 1 for simulation of dynamic PK/PD experiment). By definition, half-life of drug in the constant infusion regimen is infinity. In contrast, CMAX studies are performed with a half-life of just 4 hr. This eliminates 98% of drug by 24 hr (one-half of a life cycle) and creates large disparities between the two regimens, with respect to concentration and time. At 72 hr parasites from each cartridge are counted and the result for each dosing regimen is compared to that of untreated control. The regimen providing greater efficacy reveals the governing PK parameter. If non-specific drug binding and release from system components affect the kinetics, the apparatus is pre-incubated to block nonspecific binding and decay rates are kept as rapid as possible (typically ≤ 8 hr) within this limitation.
The experimental workflow follows a series of steps (Fig. 2). In Basic Protocol 1 the dose-response relationship of the drug is determined in a conventional 96-well microtiter plate assay. The resulting curve is used to select concentrations for the dynamic hollow fiber studies. Nonspecific binding of drug to system components is assessed in Basic Protocol 2, and Basic Protocol 3 is used to verify that the desired PK profiles are obtained in the cartridge system. These experiments are done with medium but no cells. If necessary, dose adjustments and experimental modifications are made to achieve the desired PK. These adjustments are specific to the drug being tested and may require iteration. Finally, in Basic Protocol 4 parasites are exposed to the desired AUC, deployed as a CMAX or TMIC regimen, and compared with a drug-free control. Experimental conditions are usually designed to allow direct comparison of the infusion regimen to the 96-well assay from Basic Protocol 1. Basic Protocol 4 is repeated three times to obtain statistical validation of results. Straightforward variations of Basic Protocol 4 include study of a different total dose (AUC), use of a different half-life for the peak regimen, or a total exposure time other than 72 hr.
Our apparatus uses the combination of a previously described system of bottles and pumps that generates dynamically changing drug concentrations (Blaser et al., 1985; Moore et al., 1994), and a newly-fashioned glass cartridge that supports the continuous and robust growth of malaria parasites (Bakshi et al., 2013). The hollow glass cylinder cartridge is traversed by semipermeable, macroscopically perforated dialysis tubing (Fig. 3). This creates two compartments within the cartridge (intra- and extra-tubing) and allows transmembrane movement of drugs and macromolecules. Cells settle and remain at the bottom of the glass cartridge in the extra-tubing space, which serves as the PD compartment. The intra-tubing PK compartment of each cartridge is linked to its cognate primary loop bottle by platinum-cured silicone tubing (Fig. 4). Medium with or without drug is continuously and briskly pumped around the primary loop to effect mixing within the primary loop bottle and rapid exchange between the PK and PD compartments of the cartridge. PK samples for measurement of drug concentration can be obtained from multiple locations but most important to assay are those in the PD compartment where parasites reside.
Medium with or without drug enters and exits the system via the primary loop bottle. Input bottles provide fresh medium with no drug (control and peak regimen cartridges), or with the desired constant concentration of drug (infusion regimen cartridge). Pumped at the programmed rate, these solutions drip into the primary loop bottles while fluid simultaneously drawn from the meniscus is pumped to the output bottles. For the peak regimen, medium in the primary loop is loaded with drug at peak concentration, which is then diluted at the desired half-life with drug-free input medium.
Each cartridge assembly has three associated segments of tubing that pass through the peristaltic pump: the primary loop segment exiting the cartridge (Fig. 5, purple), the segment between the input bottle and primary loop bottle (Fig. 5, green), and the segment from the primary loop bottle to the output bottle (Fig. 5, yellow). During an experiment, medium is pumped from the input bottle into the primary loop bottle. Volume in the primary loop bottle is kept constant by withdrawing fluid from the meniscus and transferring it to the output bottle. For the CMAX regimen, replenishment of drug-containing medium with drug-free medium, while keeping total volume constant, results in a controlled decrease in drug concentration in the primary loop (see Video 1). For the TMIC regimen, dilution medium contains drug at the infusion concentration, resulting in no net change in drug concentration.
The most critical flow rate in the experiment is dictated by the inner diameter of the pump tubing between the input and primary loop bottles. It governs the clearance (Cl), and hence t1/2, of drug in the CMAX regimen. A matching output rate from the primary loop bottle (and a fixed circulating volume) are achieved by using output module pump tubing whose inner diameter exceeds that of the input module, and by withdrawing fluid from the meniscus of the primary loop bottle. Relatively rapid equilibration between the primary loop bottle and cartridge is obtained by circulating medium through the primary loop significantly faster than the rate of dilution. To achieve this, primary loop pump tubing has an inner diameter 4–5 times greater than that of the input tubing. All three lines are run at the same pump setting. Actual pump tubing diameters should be chosen to fit the desired half-life and system volume of a given experiment.
For convenience and consistency, system components are grouped and classified as follows.
For purine auxotroph and obligate pyrimidine-synthesizing P. falciparum, metabolic incorporation of [3H]-hypoxanthine forms the basis of this conventional assay for the antimalarial activity of a drug against intraerythrocytic asexual parasites (Desjardins et al., 1979). The concentration that achieves a given antimalarial effectiveness (e.g., EC40 for 40% efficacy) is determined through nonlinear curve fitting of the data (Posner et al., 1997). Unless specified otherwise, all operations described below must be performed inside a biological safety cabinet (BSC).
Basic Protocols 2 and 3 describe the preliminary PK experiments, which are done without parasites. These studies ensure that the desired PK profile is actually obtained experimentally, for both the CMAX and TMIC regimens. Non-specific drug binding to hardware components is a major factor that must be accounted for when using this in vitro test system. Such binding varies based on the affinity of the compound for system components, and must be assayed every time a new compound is studied or a system component is modified. There are two aspects to the non-specific binding phenomenon: drug binding to the system and drug release from this bound pool. Both processes will affect final drug concentrations and the PKs that are achieved. As described below these experiments are most conveniently performed using radiolabeled drug as a tracer, but if this is not feasible drug concentrations may be analyzed by another method (e.g., LC-MS/MS or bioassay).
In Basic Protocol 2 the extent and kinetics of drug binding and release are assessed. Two primary loops are charged with medium containing a high or low level of drug (corresponding to the desired peak or infusion concentration, respectively) and spiked with radiolabeled tracer. To measure drug adherence, the solutions are circulated around the primary loops and samples taken from the cartridge PD compartments over 24 hr are assayed via liquid scintillation. Differences between concentrations at start and end of the experiment reveal the extent, if any, of drug adherence to system components. To measure drug release, a portion of the circulating fluid is replaced with drug- (and radioisotope-) free medium. The time-dependent appearance of radioactivity in subsequent cartridge samples reveals the extent and rate of drug release. If the difference between desired and expected results is >10%, experimental conditions must be adjusted. Knowing the extent of drug binding in the low concentration system is sufficient to finalize design of the TMIC regimen for Basic Protocol 4 However, to adjust the peak regimen both binding and release results from the high concentration conditions are required, and these more extensive modifications must be validated in Basic Protocol 3.
Meticulous sterile technique is paramount for success of these protocols. Only steps 1–10 (primary loop construction) of Basic Protocol 2 may be performed on the bench. All other steps described in Basic Protocols 2–4 must be performed in a biological safety cabinet (BSC). Do not use paper products (e.g., laboratory wipes or paper towels) in the BSC since these are known to introduce contamination. Instead, use Curity sponges (or a similar product) and 70% ethanol for cleaning the BSC and equipment.
This protocol tests, in duplicate, whether the desired peak concentration and t1/2 for the CMAX regimen are indeed obtained. Medium, with peak drug concentration and spiked with radiolabeled tracer, is loaded into the primary loop, then diluted over time with drug-free medium by means of input and output lines and bottles (see Video 1 for depiction of the dilution process). Timed samples are taken from each cartridge and quantitated by liquid scintillation counting. Plots of dpm versus time show the fall in drug concentrations over time. Comparison of the duplicate results with one another and with the desired kinetics will reveal whether further adjustments are necessary. If so, AUC equivalence must be maintained between the infusion and peak dosing regimens, and the adjusted CMAX conditions must be tested in a repeat of this Basic Protocol 3.
Note: The 4 hr default half-life for our PK/PD determinations (see Experimental Strategy) can be obtained only if drug binding is ≤ 10% or if the release half-life is ≤4.5 hr. If the release half-life determined in Basic Protocol 2 exceeds 4.5 hr, then the drug clearance rate in Basic Protocols 3 and 4 must be programmed to be longer than the non-specific release half-life. Additionally, peak concentration must be recalculated for the new half-life so as to maintain AUC equivalence.
For drugs with >10% non-specific binding, pre-incubate the system and confirm PK (For drugs with negligible non-specific binding, go directly to step 21)
The pharmacokinetic requirement for efficacy, also known as the “essential PK/PD relationship,” underpins the rational dosing of drugs and is useful for many stages of drug development (Bakshi et al., 2013). This protocol describes an experimental strategy to determine the essential PK/PD relationship of a compound against P. falciparum. It is important to note that this same experimental strategy can be used not only to study artificial kinetics, but also to model the known kinetics of a drug in animals or humans. Furthermore, though the described end point (parasite density) is taken just once (at 72 hr), many different PD endpoints may be assayed, at intervals other than 72 hr.
We strongly advise running a set of flask controls with each PK/PD run to monitor inter-experiment variation in drug susceptibility of cells. Note that while the infusion cartridge is designed to match the 96-well assay, drug susceptibility may differ between the constantly nutrient-replenished cartridge and the microtiter plate. Flasks mimic plate conditions, afford the opportunity to use the identical culture aliquoted into the cartridges, and provide comparable samples for pharmacodynamic analysis at the end of the experiment.
For simplicity, in all cartridge experiments we denote cartridge 1 as the TMIC infusion, cartridge 2 as the CMAX peak, and cartridge 3 as the control. This is critical in terms of assembly since the peak and control cartridges share the same input and output bottles.
For drugs with negligible non-specific binding, skip to step 6
A reliable and continuous supply of RBCs is essential for successfully propagating P. falciparum in the laboratory. We obtain citrate-anticoagulated O+ human blood weekly from healthy donors under an IRB-approved protocol. Donors cannot have hemoglobinopathy, G6PD deficiency, or abnormalities on routine clinical lab tests; must test negative for HIV, hepatitis B or C; may not routinely take any prescribed or over-the-counter drugs; and may not take any drug within three days of donation. For researchers without access to such a resource, RBCs may also be obtained from commercial suppliers (e.g., The Interstate Companies Laboratory, Memphis, TN). Human blood poses a biohazard; proper registration and precautions are necessary. All RBC processing must be performed in aseptic conditions within a BSC.
P. falciparum HOX was adapted from the NF54 drug-sensitive strain of parasites to survive in the high oxygen content (~19% O2) of conventional 5% CO2 incubator conditions (Bakshi et al., 2013). HOX is available from ATCC and is relatively simple to maintain. P. falciparum is a human pathogen; proper registration and precautions are necessary.
This describes staining P. falciparum with acridine orange dye and the counting RBCs and parasites using fluorescence microscopy.
Most components of the PK/PD system are custom-built. This provides a brief overview of hardware construction. Detailed instructions and diagrams are beyond the scope of this manuscript; researchers are encouraged to contact the authors directly if further information is needed.
Masterflex platinum-cured silicone tubing 96410-14 (1.6 mm i.d., 1/16″ hose barb; Cole-Parmer EW-96403-14)
Masterflex platinum-cured silicone tubing 96410-15 (4.8 mm i.d., 3/16″ hose barb; Cole Parmer EW-96410-15)
Purple-white 2-stop platinum-cured silicone tubing (2.79 mm i.d.; Idex SC0634); Orange-white 2-stop platinum-cured silicone tubing (0.64 mm i.d.; Idex SC0621); Red-red 2-stop platinum-cured silicone tubing (1.14 mm i.d.; Idex SC0625A)
|Cole-Parmer Part Number||Description|
|T-45505-08||Large Male Luer 3/16″|
|EW-45505-02||Male Luer 3/32″|
|T-45502-08||Large Female Luer 3/16″|
|EW-45502-02||Female Luer 3/32″|
|EW-06365-25||Elbow Connector 3/32″|
|EW-06365-11||Straight Connector 1/16″|
|SI-30800-12||Female Luer Cap|
|EW-30800-30||Male Luer Cap|
Qorpak bottle beaker, 60 mL (Fisher 2992601)
Glass media storage bottle with GL45 screw cap (Corning 1395-1L)
GL45 media bottle cap, low temperature orange (Pyrex 1395-45LTC)
Use distilled, deionized water (ddH2O) in all recipes and protocol steps. Individual chemicals can be purchased from Sigma unless otherwise specified.
Multidrug resistance in P. falciparum malaria has created a heightened interest in the identification and development of novel drug candidates. Various methods have been devised to judge the promise of new antimalarial drug candidates, including microtiter plate-based in vitro cytotoxicity assays (some suitable for high throughput screens) and modifications of these methods that focus on time-kill parameters (Sanz et al., 2012; Le Manach, 2013; Sherlach and Roepe, 2014 and references therein). However, most in vitro studies use fixed drug concentrations for a defined time interval, and hence do not capture the effect of dynamic fluctuations of drug concentrations in vivo. Since P. falciparum survives only in humans, in vivo models typically involve non-falciparum species that infect rodents or nonhuman primates. While these do provide dynamic drug concentrations, neither the host nor the parasite species is directly relevant. The biology and pathophysiology of infection can vary substantially from those of P. falciparum in humans, as can the metabolism and kinetics of the drug. Partially addressing this is the humanized mouse model for P. falciparum, which is costly and requires severe immunodeficiency in the animals as well as daily infusions of human RBCs (Jimenez-Diaz et al., 2009).
To overcome these major obstacles and facilitate PK/PD analysis, we developed a preclinical model system in which P. falciparum malaria parasites are exposed to dynamic drug concentrations in vitro. This system can be used to study the PK dependence of many different PD endpoints (e.g., parasite growth – including time-kill, intracellular accumulation of drug metabolites, metabolomics changes, and emergence of drug resistance), thus facilitating drug development at a number of points. Since PK profiles can be simulated without the limitations imposed by animal physiology, the PK parameter that governs efficacy can be unambiguously identified. Comparing the time- or concentration-governance (elucidated with our system) with PKs subsequently obtained in animals provides an additional rational basis for selection of lead candidates, and gives guidance for their further testing in the drug development pipeline.
The system can also be programmed to mimic known kinetics in humans. For anti-infective drugs in development, this capability has proven valuable in selecting the best dosing regimens for clinical trials (Brown et al., 2011), thus saving considerable time and expense. Moreover, using in vitro dynamic PK/PD allows the anti-infective efficacy and prevention of resistance by combination drug regimens to be evaluated in a systematic manner, an approach simply not possible in clinical trials (Drusano et al., 2014).
Before beginning the dynamic PK/PD cartridge studies described in this unit, it is important to understand the underpinning PK/PD concepts (Rowland and Tozer, 2004). Two kinetic regimens are being compared: the constant infusion held above a minimum inhibitory concentration versus the high peak concentration that is rapidly eliminated - the TMIC and CMAX regimen, respectively - in order to determine the PK parameter governing drug efficacy. For a given experiment, it is critical that the two regimens produce the same overall total drug exposure, or AUC. Adjusting for non-specific drug binding to and release from system components ensures that the desired pharmacokinetics can, in fact, be achieved. Furthermore, some drugs may not be stable in aqueous solution for the 72 hr duration of these experiments. Final verification that expected PKs are actually obtained requires quantitative analysis of drug concentrations in timed samples taken from the PD compartment, where cells reside.
In order to successfully complete Basic Protocols 2–4, several critical factors must be taken into account. The preparation and assembly of cartridges needs to be as uniform as possible. This can be challenging at first; however, Video 2 provides a visual aid and guided step-by-step instructions to achieve consistently reliable results. Construction of the apparatus for Basic Protocols 3 and 4 becomes relatively straightforward once direction of flow is understood (see Fig. 5 and Video 3). Aseptic technique throughout these experiments is of paramount importance. The considerable time, manpower, and expense of completing all four protocols is too great to risk contamination because of inadequate sterile technique.
The drug-sensitive NF54 HOX strain used in these protocols can be substituted with other strains that have not been adapted to relatively high O2 atmosphere, provided the apparatus is maintained in an incubator with microaerophilic (2–5% O2) conditions. Given the biological variation between strains, it is critical to be consistent with strain choice throughout all four protocols. To account for intrinsic variation in malaria cell culture in vitro and to obtain statistical validity, Basic Protocols 1 and 4 should be run independently at least three times.
The major mishaps in these experiments are overgrowth by contaminating microbes and failure of various system components. Our low rate of <10% cartridge contamination, despite not using antibiotics in the medium, is attributable to meticulous sterile technique, thoroughly autoclaving the system components, banning paper products (laboratory wipes, cardboard) from the BSC area, and ensuring all connections are tight. Once contaminated, everything but the glass cartridges must be discarded, and the cartridges should be soaked overnight in 3% sodium hypochlorite (household bleach).
The most common mechanical problems involve pump tubing and the glass cartridge. If the 2-stop tubing is not properly positioned in the cassettes, medium will not flow through the system correctly, affecting drug clearance and making the peak and infusion regimens incomparable. This problem can be avoided by ensuring that the placement of all 2-stop tubings is secure, and monitoring the system periodically to detect and correct tubing slippage. If tubing misalignment does occur, we advise stopping the pump, realigning the tubing within the cassette, then continuing the run. The narrow diameter tubings are also susceptible to clogging. This is minimized by the use of Albumax rather than human serum, and can further be avoided if the system is dismantled promptly and thoroughly rinsed after each run. Deformation of the 2-stop tubing after repeated use and autoclaving will lead to erroneous flow rates. We recommend discarding it after three to four uses, regardless of apparent condition.
Although uncommon, leaks do occur, usually due to an improper seal at the inlet or outlet of the cartridge. It is important to check the cartridge periodically after addition of the parasitized RBC suspension. Leakage may reflect improper assembly of the dialysis tubing, or deformities in the inlet/outlet tubing due to overuse. We recommend discarding inlet/outlet tubing after six uses regardless of apparent condition. Treating a leaking joint with 70% ethanol and tightly wrapping it in parafilm may allow successful completion of the experiment. However, in most cases, it is usually more efficient to abort the run and restart after fixing the problems listed above.
Once Basic Protocols 1–3 have been satisfactorily completed, the definitive PK/PD experiments of Basic Protocol 4 have proven quite reproducible. Nevertheless, unexpected or variable results may arise. In addition to microbial contamination and mechanical failures described above, malaria parasites are exquisitely sensitive to some plastics. Even different lots of the same product may cause toxicity. Materials listed throughout this unit have been carefully tested for compatibility with malaria parasites, and in general they represent the very few options that have proven nontoxic. However, when a new lot of tubing (in particular) is purchased or if the composition of a system component changes, we advise testing for any effect on parasite proliferation before proceeding with PK/PD experimentation. Parallel testing of drug response in flasks in Basic Protocol 4 steps 21–22 provides a basis for detecting and analyzing any unexpected PK/PD results.
In Basic Protocol 1, a dose-response curve is generated based on [3H]-hypoxanthine incorporation as a measure of antimalarial activity. In our hands, EC50 values from independent determinations generally fall within 30% of one another (Posner et al., 1997). Basic Protocol 2 evaluates interaction of the drug with the hollow fiber system components, while Basic Protocol 3 provides a basis for making (and testing) adjustments that compensate for this common effect. Ultimate validation of this method stems from assay of drug concentrations in timed PK samples. PK/PD governance is ascertained in Basic Protocol 4. The relatively simple PD endpoint we use – cell count – can be measured directly by microscopy, as described (tedious, time-consuming, somewhat subjective) or by surrogate assays e.g. [3H]-hypoxanthine incorporation or SYBR Green (Sherlach and Roepe, 2014 and references therein). In our hands and for triplicate determinations, the standard deviations in percent efficacy obtained in Basic Protocol 4 are typically within 10% of the mean.
This work is labor- and time-intensive. P. falciparum’s 48 hr life cycle mandates that these experiments, which may be completed within hours for most bacteria, require several days for malaria parasites. Assuming availability of radiolabeled tracer, no unanticipated problems, straightforward results in the drug-binding and -release studies, and support staff to prepare and breakdown the apparatus, at least six weeks of continuous experimental time are required to run a drug through all four sequential protocols, including three independent runs of Basic Protocol 4.
The 96-well microtiter plate cytotoxicity assay in Basic Protocol 1 spans five days; however, only about 6 hr of hands-on work is required. From setup to data analysis, Basic Protocol 2 and 3 can require 7–14 days, depending on the extent of drug binding and whether iterative adjustments and validations are necessary. Basic Protocol 4 requires five days to set up and run one experiment, plus time for data analysis.
Dynamics of the PK/PD system are captured by time-lapse video. Depicted is a complete rig, comprising three cartridge assemblies, the pump, and all supporting bottles and tubing. Tissue culture medium and test drug are simulated by yellow and green dye, respectively. Parasitized RBCs, normally settled on the floor of the cartridges, are not shown. During the experiment, fluid flows through all three cartridges at the same rate. Drug levels in control cartridge 3 (yellow, no drug) and in TMIC infusion cartridge 1 (light green, low drug concentration) remain constant. However, the initial high peak concentration of drug in CMAX cartridge 2 (dark green) falls dynamically over time with a half-life of 0.5 hr, and is >99% eliminated by the end of the experiment. Though dramatically different in its deployment, the total mass (dose, mg) of drug that flows through cartridges 1 and 2 is identical, as is the AUC of their concentration-time curves. Filmed with GoPro HERO 4 Silver at 0.2 sec/image and 0.5 sec lapses; edited with GroPro Studio Version 22.214.171.124. Actual filming time 5 hr, 45 min; displayed at 5 times actual; duration 03:12. No voiceover.
Step-by-step construction of a cartridge for PK/PD study of malaria parasites, including custom-made glass module, dialysis tubing, and tubing connectors. Filmed with Sony Handycam DCR-DVD650; edited with iMovie Version 10.0.3 and Pro Tools Version 12.1. Video (except hole-punching sequence at 4 times actual) is in real time, duration 05:21.
The necessary components to support three cartridges are assembled into a complete PK/PD rig, ready for the start of an experiment. Filmed with GoPro HERO 4 Silver; edited with GroPro Studio Version 126.96.36.199 and Pro Tools Version 12.1. Video is in real time, duration 09:48.
We are grateful to Zachary Wilson and Kirsten Meyer for filming and editing videos, Niall Owen McCusker for audio post-production, and Matt Shapiro for loaning a GoPro camera.
This work was supported by the NIH (grants R01AI095453, R01AI111962, and UL1TR001079), and by the Johns Hopkins Malaria Research Institute and the Bloomberg Family Foundation.
Conflict of Interest
No conflict of interest for any of the authors.