Hollow‐Fiber Methodology for Pharmacokinetic/Pharmacodynamic Studies of Antimalarial Compounds

Emily Caton1, Elizabeth Nenortas1, Rahul P. Bakshi1, Theresa A. Shapiro1

1 Division of Clinical Pharmacology, Departments of Medicine and of Pharmacology and Molecular Sciences, and The Johns Hopkins Malaria Research Institute, The Johns Hopkins University, Maryland
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch150194
Online Posting Date:  March, 2016
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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 the 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. © 2016 by John Wiley & Sons, Inc.

Keywords: Plasmodium falciparum; pharmacokinetics; pharmacodynamics; dynamic PK/PD; hollow‐fiber cartridge system; drug development; malaria

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Antimalarial Dose‐Response by Microtiter Plate Assay
  • Basic Protocol 2: Assessment of Nonspecific Drug Binding and Release
  • Basic Protocol 3: Validation of Dynamic PK for CMAX Regimen
  • Basic Protocol 4: Dynamic Hollow‐Fiber Studies for Antimalarial PK/PD
  • Support Protocol 1: Preparation of Washed RBCs
  • Support Protocol 2: Maintaining P. falciparum HOX in Continuous Culture
  • Support Protocol 3: Measuring Parasitemia
  • Support Protocol 4: System Components and Construction
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Antimalarial Dose‐Response by Microtiter Plate Assay

  • Drug stock solutions (typically in DMSO or water)
  • P. falciparum medium (see recipe)
  • Uninfected erythrocytes (red blood cells, RBCs)
  • P. falciparum HOX (ATCC)
  • [3H(G)]Hypoxanthine monohydrochloride (1 mCi/ml, 14 Ci/mmol, 574 GBq/mmol; PerkinElmer, cat. no. NET0177)
  • 96‐well microtiter plate, flat‐bottomed wells (Costar 3596)
  • Humidified incubation chamber for 96‐well plates (CBS Scientific, cat. no. M‐312)
  • 50‐ml sterile disposable conical tubes (Sarstedt, Part No. 62‐557‐205)
  • GF/C glass fiber filters (Brandel Inc.)
  • Cell harvester (Brandel Inc., Model MB48)
  • Heat lamp (optional)
  • Liquid scintillation counter
  • Microsoft Excel, GraphPad Prism, or similar software for nonlinear curve fitting
  • Additional reagents and equipment for maintaining P. falciparum in continuous culture ( protocol 6) and determining parasitemia ( protocol 7)

Basic Protocol 2: Assessment of Nonspecific Drug Binding and Release

  • Radiolabeled drug tracer (≥1 μCi/μl; 10 μl/50 ml medium is sufficient for the experiments described below; tracer should contribute ≤1% of total drug mass)
  • 70% ethanol
  • P. falciparum medium (see recipe)
  • Scintillation fluid
  • Spectra/Por 7 dialysis tubing: 28 mm flat width, 50 kD MWCO (Spectrum Laboratories, cat. no. 132129)
  • Aluminum foil
  • 31‐G syringe needles
  • Sewing needle and thread
  • Glass cartridges (custom from Adams & Chittenden Scientific Glass)
  • Injection ports (Interlink system injection site; Baxter, cat. no. 2N3399)
  • Slide clips (Qosina, cat. no. 95868)
  • 18‐G blunt reusable syringe needles
  • 50‐ml conical tubes (e.g., Corning Falcon)
  • Curity 4‐in. × 4‐in. All Purpose Sponges (Covidien)
  • Sterile syringe filters, 0.22‐μm (Olympus)
  • Ismatech IP 12‐channel standard‐speed digital dispensing pump (Cole Parmer, cat. no. EW‐78001‐22)
  • 1‐ml and 20‐ml syringes
  • Scintillation vials
  • Microsoft Excel, GraphPad Prism, or similar software for nonlinear curve fitting
  • Additional reagents and equipment for dose‐response microtiter assay ( protocol 1) and construction of the PK/PD system ( protocol 8)

Basic Protocol 3: Validation of Dynamic PK for CMAX Regimen

  • 25‐cm2 (T‐25) vented flasks
  • Incubator shelf (which conveniently serves as a tray for transferring apparatus between BSC and incubator)
  • Additional reagents and equipment for PK/PD (see protocol 1Basic Protocols 1 to protocol 33 and protocol 5Support Protocols 1 to protocol 84)

Basic Protocol 4: Dynamic Hollow‐Fiber Studies for Antimalarial PK/PD

  • Human whole blood
  • Incomplete medium (see recipe)
  • 15‐ml sterile capped conical centrifuge tubes (Sarstedt, cat. no.. 62‐554‐205)
  • Tabletop centrifuge

Support Protocol 1: Preparation of Washed RBCs

  • Washed RBCs at 50% hematocrit
  • P. falciparum medium (see recipe)
  • P. falciparum HOX (ATCC)
  • 25‐cm2 (T‐25) vented tissue culture flasks
  • Additional reagents and equipment for measuring parasitemia ( protocol 7)

Support Protocol 2: Maintaining P. falciparum HOX in Continuous Culture

  • P. falciparum HOX culture to be counted
  • 100% methanol
  • 100 μg/ml acridine orange (Sigma) in 1× phosphate‐buffered saline pH 7.4 (Quality Biological, Inc.)
  • Glass microscope slides (Fisher, cat. no. 12‐550‐15)
  • Microscope coverglass (Fisher, cat. no. 12‐542‐B)
  • Slide storage box
  • Fluorescence microscope or light microscope fitted with Paralens Illuminator

Support Protocol 3: Measuring Parasitemia

  • Standard connecting tubing: Masterflex platinum‐cured silicone tubing 96410‐14 (1.6 mm i.d., 1/16‐in. hose barb; Cole‐Parmer, cat. no. EW‐96403‐14)
  • Cartridge tubing (for cartridge sampling ports and inlet/outlet): Masterflex platinum‐cured silicone tubing 96410‐15 (4.8 mm i.d., 3/16‐in. hose barb; Cole Parmer, cat. no. EW‐96410‐15)
  • 2‐stop tubing: 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, cat. no. SC0621); red‐red 2‐stop platinum‐cured silicone tubing (1.14 mm i.d.; Idex, cat. no. SC0625A)
  • Fittings:
    • Large Male Luer, 3/16‐in. (Cole Parmer, cat. no. T‐45505‐08)
    • Male Luer 3/32‐in. (Cole Parmer, cat. no. EW‐45505‐02)
    • Large Female Luer 3/16‐in. (Cole Parmer, cat. no. T‐45502‐08)
    • Female Luer 3/32‐in. (Cole Parmer, cat. no. EW‐45502‐02)
    • Elbow Connector 3/32‐in. (Cole Parmer, cat. no. EW‐06365‐25)
    • Straight Connector 1/16‐in. (Cole Parmer, cat. no. EW‐06365‐11)
    • Female Luer Cap (Cole Parmer, cat. no. SI‐30800‐12)
    • Male Luer Cap (Cole Parmer, cat. no. EW‐30800‐30)
  • Primary loop bottle: Qorpak bottle beaker, 60 ml (Fisher, cat. no. 2992601)
  • Input/output bottle: Glass media storage bottle with GL45 screw cap (Corning, cat. no. 1395‐1 liter)
  • Input/output bottle caps: GL45 media bottle cap, low temperature, orange (Corning, cat. no. 1395‐45LTC)
  • DAP Auto/Marine Sealant 100% RTV Silicone (Amazon.com or local hardware store)
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