Overview of Pharmacokinetics

Lisa D. Aimone1, Inés A.M. de Lannoy2

1 Teva Pharmaceuticals, Malvern, 2 InterVivo Solutions, Toronto, Ontario
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.1
DOI:  10.1002/0471141755.ph0701s66
Online Posting Date:  September, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

In addition to having selective potency against the molecular target(s), a compound must be able to reach its intended site of action in vivo in sufficient quantity and for the appropriate duration of time to exert a biological effect. The fate of a compound after in vivo administration depends upon its absorption, distribution, metabolism, and excretion (ADME), as well as its target residence time. The concentration of the compound in the blood, plasma, and other tissues represents the sum of all of these processes. Described in this unit are protocols for administering a compound by various routes to rats and for collecting the appropriate samples to determine the pharmacokinetics profile. The basic terms used in pharmacokinetics studies are defined, and representative examples are given to illustrate important variables to consider. Curr. Protoc. Pharmacol. 66:7.1.1‐7.1.31. © 2014 by John Wiley & Sons, Inc.

Keywords: pharmacokinetics parameters; in vivo routes of administration; blood and tissue collection; bioavailability; half‐life; mass balance

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Compound Administration in Rats
  • Support Protocol 1: Isoflurane Anesthesia of the Rat
  • Basic Protocol 2: Blood Collection in Rats
  • Basic Protocol 3: Excreta Collection in Rats
  • Basic Protocol 4: Tissue Collection in Rats
  • Support Protocol 2: Sample Processing: Determination of Radiolabeled Compound
  • Support Protocol 3: Sample Processing: Determination of Nonradiolabeled Compounds
  • Support Protocol 4: Calculations for Radiolabeled Compounds
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Compound Administration in Rats

  Materials
  • Formulated test compound (see unit 7.3 for overview on formulation)
  • Male or female albino rats (∼250 and 200 g, respectively), with or without jugular vein and carotid artery catheters (catheterized preferably in house or purchased from Charles River Labs, Harlan Laboratories, or Hilltop Lab Animals)
  • Warm soap solution or alcohol pads
  • Sterile, heparinized physiological (0.9%) saline
Appropriate‐sized needles:
  • 23‐ or 25‐G × 1.5 in. (∼3.75 cm) for i.v., s.c., and i.p. administration
  • 25‐ to 26‐G × 0.5‐in. (∼1.75 cm) for i.m. administration
  • 15‐ to 18‐G × 3‐in. (∼7.5 cm) straight or curved gavage needle with 2.25 mm bulb for p.o. administration
Appropriate‐sized single‐use polypropylene syringes:
  • 1 ml for i.v., s.c., and i.m. administration
  • 3 ml for i.p. and p.o. administration
  • Balance accurate to 0.001 g
  • Broome‐style or other appropriate rodent restrainer (VWR or Fisher)
  • Disposable gloves
  • Gauze
  • Heat lamp
  • Padded (i.e., gauze‐wrapped) paper clip (optional)
  • Electric clippers (optional)
  • Additional reagents and equipment for isoflurane anesthesia of the rat (see protocol 2)

Support Protocol 1: Isoflurane Anesthesia of the Rat

  Materials
  • Rats
  • Isoflurane
  • Oxygen cylinder and regulator
  • Ophthalmic ointment (e.g., Tear Naturale P.M., Alcon)
  • 1‐gallon (∼4‐liter) glass jar with lid, separated vertically into two compartments by a plastic mesh screen
  • 50‐ml conical polypropylene centrifuge tube (e.g., BD Falcon)
  • Gauze
  • Induction chamber (e.g., EZ‐175 Biovac Chamber with EZ‐255 BioVac Filter; E‐Z Anesthesia, http://www.ezanesthesia.com/)
  • Isoflurane vaporizer (e.g., 53‐T3ISO TEC III Vaporizer; Benson Medical Industries)
  • Isoflurane anesthesia machine (e.g., VMR Table Top Anesthetic Machine; Benson Medical Industries)
  • Rebreathing circuit with nose cone (e.g., Universal “F” rebreathing circuit 50522; Benson Medical Industries)
  • Heating pad with temperature control
  • Heat lamp

Basic Protocol 2: Blood Collection in Rats

  Materials
  • Male or female (∼250 and 200 g, respectively) jugular vein and/or carotid artery catheterized albino rats (catheterized in‐house or commercially available from, e.g., Charles River Labs, Hilltop Lab Animals, or Harlan Laboratories) or non‐catheterized albino rats and appropriate catheters [e.g., short jugular catheter (blue), BASi, cat. no. CX‐20115, and carotid catheter (red), BASi, cat. no. CX‐20125; http://www.basinc.com/]
  • 0.9% saline (NaCl), sterile
  • Heparinized 0.9% saline: 400 IU/ml heparin in sterile 0.9% NaCl
  • Warm soap solution or alcohol pads
  • 1‐ml polypropylene syringes furnished with blunt‐tip needles that fit snugly into the end of the catheter, e.g., 21‐G × 1‐in (∼2.5 cm)
  • Hemostats, padded (i.e., gauze‐wrapped)
  • Heparin‐ (sodium or lithium) or K 2EDTA‐coated Microtainer tubes: purchase or rinse plain 1.5‐ml microcentrifuge tubes with heparinized saline (see above) and let dry
  • Broome‐style or other appropriate rodent restrainer
  • Gauze pads
  • Cotton‐tipped applicators
  • Surgical instruments including scissors and hemostat
  • Heparin‐ (sodium or lithium) or K 2EDTA‐coated Vacutainer tubes
  • Heparinized 10‐ml syringe with 21‐G, 1‐in (∼2.5‐cm) needle
  • Additional reagents and equipment for anesthesia of rats ( protocol 2)

Basic Protocol 3: Excreta Collection in Rats

  Materials
  • Male or female albino rats (∼250 and 200 g, respectively)
  • 50:50 (v/v) methanol/water
  • Bile duct–cannulated rats (cannulated in‐house or from a commercial source)
  • Sodium taurocholate solution (see recipe)
  • Metabolic cages and containers (Nalgene or equivalent)
  • Balance accurate to 0.001 g
  • Ring stand and clamp
  • PE 60 polyethylene tubing, 0.76‐mm i.d., 1.22‐mm o.d. (VWR or Fisher)
  • Tether and dual‐port swivel apparatus (Instech Labs)
  • 3‐ and 5‐ml syringes with 21‐G blunt needles
  • 21‐G stainless steel tubing
  • Syringe pump and 50‐ml syringe
  • Animal restraint jackets (Instech Labs)
  • Additional reagents and equipment for anesthesia of the rat (see protocol 2), compound administration in the rat ( protocol 1), and sample processing to determine radiolabeled compound ( protocol 6)

Basic Protocol 4: Tissue Collection in Rats

  Materials
  • Rat administered test compound (see protocol 1)
  • Wash and rinse solutions for instruments (standard dishwashing liquid and deionized water)
  • 0.9% NaCl (optional)
  • 1‐ml syringe with 27‐G needle
  • 500‐ml beakers for solid waste
  • Sharps container
  • Dissecting instruments including:
  • Hemostats: 3.5‐ (∼8.75‐cm) or 5‐in. (∼12.5‐cm) straight and 3.5‐ or 5‐in. curved
  • Forceps: 4‐in. (∼10‐cm) ring‐tipped, 4‐in. with straight serrated jaws, and 4‐in. with curved serrated jaws
  • Scissors: 4.5‐in. (∼11.25‐cm) straight with 25‐mm blades, 4‐in. (∼10‐cm) straight with probe points and 26‐mm blades, and 5.5‐in. (∼13.75) straight with sharp/blunt tips
  • Scalpel
  • Spatula (standard weighing spatula with rounded and pointed ends)
  • Gauze
  • Petri dishes
  • Specimen cups with caps
  • Electric clippers
  • Additional reagents and equipment for anesthesia (see protocol 2) and exsanguination of the rat (see protocol 3, steps 2c to 4c)

Support Protocol 2: Sample Processing: Determination of Radiolabeled Compound

  Materials
  • Blood (see protocol 3), excreta (see protocol 4), and/or tissue samples (see protocol 5) from compound‐treated rat (see protocol 1)
  • Liquid scintillation cocktail
  • 5 M KOH in 1:1 (v/v) methanol/H 2O
  • 6 M HCl
  • Combustocones for Perkin Elmer Sample Oxidizer Model 307
  • Combustopads for Perkin Elmer Sample Oxidizer Model 307
  • Refrigerated centrifuge
  • Cryovial with cap, or equivalent‐sized polypropylene container
  • Glass scintillation vials
  • Balance accurate to 0.001 g
  • Homogenizer and probe (e.g., Polytron, Brinkmann)
  • Combustor (Perkin Elmer Sample Oxidizer Model 307)

Support Protocol 3: Sample Processing: Determination of Nonradiolabeled Compounds

  Materials
  • Acetonitrile , HPLC grade, 4°C
  • Methanol, HPLC grade, 4°C
  • Appropriate internal standard to be used for LC‐MS/MS
  • Phosphate‐buffered saline, pH 7.4 (PBS; appendix 2A), 4°C
  • Refrigerated centrifuge, 4°C
  • LC‐MS/MS instrument with appropriate autosampler vials
  • 1.5‐, 15‐, and 50‐ml polypropylene centrifuge tubes
  • Homogenizer and probe (e.g., Polytron, Brinkmann)
  • Disposable probes (TissueRuptor, Qiagen)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Abdel‐Rahman, S.M. and Kauffman, R.E. 2004. The integration of pharmacokinetics and pharmacodynamics: Understanding dose‐response. Annu. Rev. Pharmacol. Toxicol. 44:111‐36.
  Copeland, R.A. 2013. Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists, 2nd Ed. Wiley: Hoboken, N.J.
  Diehl, K.‐H., Hull, R., Morton, D., Pfister, R., Rabemampianina, Y., Smith, D., Vidal, J.‐M., and van de Vortenbosch, C. 2001. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J. Appl. Toxicol. 21:15‐23.
  Fan, J., and de Lannoy, I.A.M. 2014. Pharmacokinetics. Biochem. Pharmacol. 87:93‐120.
  Herman, J.L. 2002. Generic method for on‐line extraction of drug substances in the presence of biological matrices using turbulent flow chromatography. Rapid Commun. Mass Spectrom. 16:421‐426.
  Hodgson, J. 2001. ADMET—turning chemicals into drugs. Nat. Biotechnol. 19:722‐726.
  Hosea, N.A. and Jones, H.M. 2013. Predicting pharmacokinetic profiles using in silico derived parameters. Mol. Pharmaceut. 10:1207‐1215.
  Kwon, Y. 2001. Handbook of Essential Pharmacokinetics, Pharmacodynamics and Drug Metabolism for Industrial Scientists. Kluwer Academic/Plenum Publishers, New York.
  Leeson, P. 2012. Drug discovery: Chemical beauty contest. Nature 481:455‐456.
  Lipinski, C.A., Lombardo, F., Dominy, B.W., and Feeney, P. 2001. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development setting. Adv. Drug Deliv. Rev. 46:3‐26.
  Popesko, P., Rajtova, V., and Horak, J. (eds.). 1992. Anatomy of Small Laboratory Animals, Vol. II. Wolfe Publishing, London.
  Tozer, T.N. and Rowland, M. 2006. Introduction to Pharmacokinetics and Pharmacodynamics: The Quantitative Basis of Drug Therapy. Lippincott Williams & Wilkins, Baltimore, Maryland.
  Waynforth, H.B. and Flecknell, P.A. 1992. Experimental and Surgical Technique in the Rat. Academic Press, London.
  Wingerd, B.D. 1988. Rat Dissection Manual. The Johns Hopkins University Press, Baltimore, Maryland.
Internet Resources
  http://www.pharsight.com
  Pharmacokinetic and pharmacodynamic modeling software.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library