Pharmacokinetic Study Methods
Animals including dogs are frequently used for pharmacokinetics (PK) and “absorption, distribution, metabolism, and excretion” (ADME) studies. These studies try to mimic what might happen when a human is given a medication, and what dose of medication will be effective while avoiding toxicity.
However, physiological and genetic differences between humans and other animals mean that results from these studies do not reflect potential human responses, putting people and animals at risk.1,2 Human blood volume, metabolic enzyme profiles,3 barrier permeability, gastric environment, clearance,4 and other factors differ substantially from other animals. In addition to these known species differences, the animals experience pain and distress that could further impact the results of such studies.
For example, drugs are often administered by gavage, which involves placing a long plastic tube down an animal’s throat into their stomach, through which the drug is administered. The animal is restrained, and there is clear evidence that the restraint and gavage administration results in rapid and significant physiological distress. Gavage also often causes local irritation and damage to the esophageal tissue; some animals have died from a ruptured esophagus.5 Stress, in turn, can drastically affect the absorption, metabolism, and excretion of chemicals.6 In dogs, stress can also affect the animal’s “gastric emptying” rate, potentially keeping drugs in the stomach longer than would normally occur.7,8
There are a variety of sophisticated methods by which ADME and dosing levels can be assessed, and examples are described below. Taken together, these techniques can provide a more human-relevant picture of how a drug is likely to behave in the human body, and what dose levels should be considered for clinical trials.9
Microdosing10 is a preclinical safety testing technique for pharmaceuticals that involves highly controlled trials of new drugs in humans, using ultralow doses tagged with a label. The technique allows researchers to get accurate information on the movement of the drug as it is absorbed and metabolized in the human body, which offers a much more accurate picture than such studies in animals. The ultralow doses ensure the trials will be safe.
Microphysiological systems, sometimes called “human-on-a-chip” systems, are truly revolutionizing medicine.11 A marriage of engineering and advanced biology, these flexible systems contain small reservoirs of cells linked by fluid-containing channels on small silicone or polymer chips. Other, larger systems with dishes of cells linked by tubes are also available. The systems allow for the linkage of different “organs”—represented by the different cell types—linked by blood vessels.12 The flexibility of the systems allows investigators to study general metabolism and distribution of drugs with many different organs or to link only two organs—for example lungs and liver—to investigate specific research questions.13
While the systems can be used to test the toxicity of drugs, they are particularly useful for PK and ADME testing, 14,15 because they can mimic the entire human physiological system, taking into account intercellular and interorgan communication. Major drug companies like Sanofi and AstraZeneca are using systems developed by HuREL16 and Harvard’s Wyss Institute,17 respectively, to test new drugs.
Human Cell-Based Methods
Cultures of epithelial cells from the body’s barriers—the skin,18 intestine,19 blood/brain, 20 and blood/lung,21 are available and used extensively to estimate how much of a drug is absorbed into the body.
Once a drug is absorbed, it is taken up by the blood and, in most cases, finds its way to the liver, known as the body’s “detox center.” In the liver, metabolic enzymes break up drugs and chemicals, often detoxifying them. Therefore, in vitro liver cells, 3-D “spheroids,” and lab-grown tissues are an important tool. Numerous companies offer cryopreserved primary hepatocytes,22 HepaRG cell lines,23 3-D liver tissue models,24 and even bioprinted25 liver “strips” to assess how humans might metabolize a drug. A recent study found that bioprinted liver strips created by San Diego-based Organovo could be used for as long as 40 days, making them useful for repeated-dose studies.26
Recent advances in computing power have led to a host of in silico tools to help predict the ADME and PK properties of a drug. Several databases are available that allow the comparison of new drugs with existing drug profiles.27 Computers can be programmed to analyze vast amounts of data and create predictive algorithms and models based on a drug’s chemical structure.28 Finally, “virtual” livers are being created that can model what might happen in a real human liver when exposed to certain drugs or chemicals.29 Besides speed, a major advantage of computational models is that they can take variation between individual people into account, which is impossible with animals.30
1 Giorgi M, Portela DA, Breghi G, et al. Pharmacokinetics and pharmacodynamics of zolpidem after oral administration of a single dose in dogs. Am J Vet Res. 2012;73:1650-1656.
2 Schmitz A, Thormann W, Moessner L, et al. Enantioselective CE analysis of hepatic ketamine metabolism in different species in vitro. Electrophoresis. 2010 May;31(9):1506-16.
3 Martignoni M, Groothuis GM, de Kanter R. Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition and induction. Expert Opin Drug Metab Toxicol. 2006 Dec;2(6):875-94.
4 Grime K, Paine SW. Species differences in biliary clearance and possible relevance of hepatic uptake and efflux transporters involvement. Drug Metab Dispos. 2013 Feb;41(2):372-8.
5 Turner PV, Brabb T, Pekow C, et al. Administration of substances to laboratory animals: routes of administration and factors to consider. J Am Assoc Lab Anim Sci. 2011 Sep;50(5):600-13.
6 Vandenberg LM, Welshons WV, vom Saal FS, et al. Should oral gavage be abandoned in toxicity testing of endocrine disruptors? Environ Health. 2014; 13:46.
7 Gué M, Peeters T, Depoortere I, et al. Stress-induced changes in gastric emptying, postprandial motility, and plasma gut hormone levels in dogs. Gastroenterology. 1989 Nov;97(5):1101-7.
8 Mistiaen W, Blockx P, Van Hee R, et al. The effect of stress on gastric emptying rate measured with a radionuclide tracer. Hepatogastroenterology. 2002 Sep-Oct;49(47):1457-60.
9 Cyprotex website. http://www.cyprotex.com/admepk/. Accessed November 7, 2014
10 Tewari T, Mukherjee S. Microdosing: Concept, Application and Relevance. Perspect Clin Res. 2010 Apr-Jun; 1(2): 61–63.
11 Human Organs-on-Chips: Tailoring Testing and Treatments. Good Medicine. Physicians Committee for Responsible Medicine website. Autumn 2014 Vol. XXIII, No. 4. Accessed November 7, 2014
12 Ghaemmaghami AM, Hancock MJ, Harrington H, et al. Biomimetic tissues on a chip for drug discovery. Drug Discov Today. 2012 Feb;17(3-4):173-81.
13 National Center for Advancing Translational Sciences website. http://www.ncats.nih.gov/research/reengineering/tissue-chip/tissue-chip.html. Accessed November 7, 2014
14 Bhatia SN, Ingber DE. Microfluidic organs-on-chips. Nat Biotechnol. 2014 Aug;32(8):760-72.
15 Maguire TJ, Novik E, Chao P, et al. Design and application of microfluidic systems for in vitro pharmacokinetic evaluation of drug candidates. Curr Drug Metab. 2009 Dec;10(10):1192-9.
16 Hurel website. http://hurelcorp.com. Accessed November 7, 2014
17 Wyss Institute website. http://wyss.harvard.edu. Accessed November 7, 2014
18 Dermal Technology Laboratory website. http://www.dermaltechnology.com/services/. Accessed November 7, 2014
19 Kim HJ, Huh D, Hamilton G, et al. Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow. Lab Chip. 2012 Jun 21;12(12):2165-74.
20 Hatherell K, Couraud PO, Romero IA, et al. Development of a three-dimensional, all-human in vitro model of the blood-brain barrier using mono-, co-, and tri-cultivation Transwell models. J Neurosci Methods. 2011 Aug 15;199(2):223-9.
21 Rothen-Rutishauser B, Blank F, Mühlfeld C, et al. In vitro models of the human epithelial airway barrier to study the toxic potential of particulate matter. Expert Opin Drug Metab Toxicol. 2008 Aug;4(8):1075-89.
22 Life Technologies website. http://www.lifetechnologies.com/us/en/home/life-science/drug-discovery/adme-tox/gibco-hepatocytes.html?cid=fl-hepatocytes. Accessed November 7, 2014
23 Marion MJ, Hantz O, Durantel D. The HepaRG cell line: biological properties and relevance as a tool for cell biology, drug metabolism, and virology studies. Methods Mol Biol. 2010;640:261-72.
24 Liu Tsang V, Chen AA, Cho LM, et al. Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels. FASEB J. 2007 Mar;21(3):790-801.
25 Murphy SV, Atala A. 3D bioprinting of tissues and organs. Nat Biotechnol. 2014 Aug;32(8):773-85.
26 Organovo website. http://www.organovo.com/tissues-services/3d-human-tissue-models-services-research/tissue-models/3d-human-liver-tissue-model. Accessed November 7, 2014
27 Law V, Knox C, Djoumbou Y, et al. DrugBank 4.0: shedding new light on drug metabolism. Nucleic Acids Res. 2014 Jan;42(Database issue):D1091-7.
28 Hsiao YW, Fagerholm U, Norinder U. In silico categorization of in vivo intrinsic clearance using machine learning. Mol Pharm. 2013 Apr 1;10(4):1318-21.
29 National Center for Computational Toxicology website. http://www.epa.gov/ncct/virtual_liver/. Accessed November 7, 2014
30 SimCyp website. http://www.simcyp.com. Accessed November 7, 2014