Ussing Chambers - A short hop from frog skin to human tissues. By David Arthur
In modern society we don’t like to talk much about the inner workings of our bowels, it’s still a taboo in polite society, but here at Biopta we talk about it almost every day. That’s because an understanding of gastrointestinal behaviour is so important to the successful development of a new drug, from its oral delivery and absorption to GI metabolism and the potential for drug-drug interactions.
Thanks to Hans Ussing, who invented the Ussing Chamber in the 1950’s, and its recent miniaturisation for use with small sections of GI mucosa we now have the ability to assess new compounds in the most relevant and predictive model available – fresh human tissue.
Such tests allow assessment of drug permeability to try and predict clinical absorption, monitor drug effects on ion channel function or to look at different transport systems and possible interactions with other compounds.
Hans Ussing: a short hop from frogs to humans
Ussing’s invention came from the observation that frogs can absorb water into their body without actually drinking it. He surmised that they must somehow absorb the water through their skin and this led him to design the Ussing chamber to test his hypothesis. He set up a system with some frog skin placed between two fluid-filled glass chambers. In each chamber were electrodes able to measure the voltage on either side of the skin. Ussing established that frogs absorb sodium ions through their skin, thereby drawing water through the skin by osmosis. Ussing achieved this by measuring the potential difference (voltage) across the membrane while simultaneously injecting current into the system; by determining the amount of current required to cancel out the voltage, he could effectively measure the net flow of ions across the membrane.
His work gave scientists the means to study almost any polarised membrane and the movement of ions across tissue. The human GI shares some similarities to frog skin, for example, we absorb sodium ions and the electrochemical gradient leads to water absorption. The GI tract can also secrete chloride ions, which causes water to be secreted. This process of sodium absorption and chloride secretion is very important and any interruption can have very dramatic and potentially fatal results. For example, infection by cholera bacteria and subsequent release of the toxin puts the chloride channels into a “hyper” secretive state, resulting in a large volume of water and salts being secreted into the intestine causing dysentery for the patient, which if untreated can be fatal. The Ussing chamber affords a sensitive measurement of current flow; compounds to treat hyper-secretion can be readily assessed.
GI Adverse Effects: A Major Problem for Clinical Development
A major reason for clinical problems is poor patient compliance due to GI side-effects1; for example, many commonly used drugs such as metformin, cancer therapies, Alzheimer’s treatments and NSAIDs cause GI effects that to the patient often lead to them abandoning treatment or failing to comply with a clinically effective dosing regimen. For example, 56% of patients receiving the molecular therapy erlatinib had diarrhea, compared with only 19% of the placebo group 2. The Ussing chamber, when used with fresh human small intestine or colon tissues, is a sensitive way to predict such GI disturbances.
Permeability and transporters
A further cause of drug failure is poor bioavailability. Using fresh human tissue in the Ussing system allows us to study a compound’s permeability across the gut mucosa and can give a prediction of the clinical fraction absorbed. Bioavailability of course involves a number of other factors such as drug solubility and first-pass metabolism, but the key factor in oral absorption is permeability across the GI mucosa. Fresh human small intestine (which is the target for absorption of most drugs) has the relevant expression of transporters, which is lacking in animal models or in human cell line systems such as the CaCo2 cell line, which is a colon cancer cell type. The Ussing chamber method therefore allows us to investigate whether a compound is a substrate of an efflux transporter such as Pgp and whether any drug-drug interactions are likely.
Perhaps there is something true about having to kiss a few frogs to find a prince; OK, so Hans Ussing didn’t need to kiss any frogs (as far as we know) but he did create a technique that is now showing its true value as a means to truly predict the behaviour of drugs in human tissues.
1. Medscape Education Article. http://www.medscape.org/viewarticle/571554_2. Gastrointestinal and Hepatic Adverse Effects of Molecular-Targeted Agents: Diarrhea
2. Shepherd FA et al. (2005) Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 353: 123-132