While in vitro studies are becoming more accurate and the resulting preclinical data more likely to predict the clinical activity of a drug, in vivo studies (studies in animals) are still vital for the assessment of pharmacokinetics and drug metabolism and essential for approval of drugs and devices by regulatory bodies. We all know that drug development is taking longer and becoming more costly. Though this is mostly driven by the clinical phases, any technology that can speed up individual steps of the preclinical stage and cut the costs is invaluable.
Over the past few decades, concern for animal welfare in drug and device testing has (rightly) increased, with advocates looking to reduce the stress that animals are under during studies and also cut the numbers of animals required for each study. Handling animals to take samples causes them stress, and restraining them during a study also causes distress and modifies their behavioral patterns. As well as having animal welfare implications, this has a pragmatic impact, as these changes could have an impact on the final results of the study. Automated in vivo sampling systems can have an impact on all of these variables.
In automated systems, the animals wear harnesses that secure sampling, drug delivery catheters, and data-generating devices. For example, in BASi’s Culex Automated In Vivo Sampling System, the harnesses are connected to sensors that track the animal’s movements. These drive motors that rotate the floor to counter the animal’s movement, allowing the animals to move freely without tangling the lines. Because of this rotational movement, the Culex small animal system is sometimes (affectionately) referred to as the “Raturn,” and the large animal version the “Pigturn.”
“The animals recover quickly from surgery to implant the catheters, and the behavior of the animals connected to the Culex system seems the same as ‘normal’ animals. They just don’t seem aware of anything unusual,” says David Hopper, director of toxicology at BASi.
The key advantage of the automated systems is that they reduce the stress, as technicians do not need to handle animals to take any samples, and even the best socialized and trained animals will become stressed with repeated sampling. “Stress can make major differences to results of trials,” says Robyn McCain, manager and biopharmaceutics technician at Purdue Translational Pharmacology Facility, Purdue University. “We have assessed this by administering the same compound via an automated system with a gastric catheter vs. traditional oral gavage [introduction of material into the stomach by a tube] in rodents. We looked at the differences in drug absorption using blood samples. The plasma levels showed a difference in the rate of absorption, and we theorize this is due to the blood supply to the gut becoming altered because of the ‘fight-or-flight’ response during oral gavage. This could have had a major impact on the outcome of an oral metabolism study.”
In a study of 16 pigs sampled using either automated blood sampling, automated blood sampling with sounds and images of manual sampling, or manual sampling, animals sampled with the automated system alone had lower levels of cortisol and norepinephrine. The cortisol levels for animals sampled manually were twice as high and norepinephrine levels were three times as high, indicating much higher levels of stress.
Automated systems can sample a number of different types of body fluid from the animal, including blood, urine, bile, metabolites, dialysates and others, as well as collecting feces.
“In traditional in vivo studies, samples of different body fluids would be taken from different animals studied in parallel, increasing the numbers of animals used and adding in variability between animals. Because automated systems can take different samples from the same animal simultaneously, as well as monitoring vital signs such as heart rate, this makes the results more consistent and much more meaningful and saves time and decreases the number of animals needed, as additional studies do not need to be run sequentially,” says Hopper.
As well as taking samples, the automated systems can administer study drugs automatically on programmed schedules through a catheter by various routes, including intravenous infusion. While in the harness, researchers can still handle the animals to administer injections or oral gavage — though this will obviously add in some stress, the overall levels are still lower than for animals that have to be captured for sampling on a regular basis.
The automated systems increase convenience for the researchers, as they can keep samples cool within the sampling system. As an additional advantage, the volumes of blood extracted from the animals are smaller than those taken manually, which is better for the animal’s health.
“The next step for systems like Culex is incorporating dry blood spot (DBS) sample collection — this removes the need for refrigeration and will make shipping of blood samples cheaper, quicker, and safer,” says Hopper. BASi is currently conducting investigative studies comparing manually spotted DBS sampling vs. traditional manual sampling and the automated spotting of samples directly on proprietary Culex DBS cards.
Automated blood sampling also increases efficiency, as members of staff are not needed around the clock to take blood samples. The combination of this, and the reduction in the number of animals, helps to reduce the overall costs of preclinical drug and device development.
Automated Sampling At Purdue
Pigs are very important for pharmacokinetic, pharmacodynamic, and toxicology studies as they are physiologically similar to humans. Purdue University uses Culex-L, the large animal version of BASi’s Culex Automated In Vivo Sampling System, for its studies of pigs.
“The Culex Automated In Vivo Sampling System was developed by BASi in the 1990s,” says Hopper. “The system was named after a genus of mosquito found in the eastern United States, an organism that has evolved to take small samples of blood and store them without clotting. Purdue was very instrumental in the development of the large animal version.” Purdue’s Culex-L has been in operation for two years, and the Purdue Translational Pharmacology Facility team uses it for both academic and commercial studies.
“Some of the pigs available for us to use are known as the Ossabaw swine. These are from an isolated colony that carry the genes for metabolic syndrome and naturally develop type II diabetes when kept on a high-fat diet. These are ideal for diabetes and obesity studies, including gastric bypass, and for atherosclerosis,” says McCain.
McCain trains the pigs and makes them used to handling before beginning a study, such as teaching them to sit (with only a little bribery from marshmallows, a particular porcine favorite!). However, like rodents and other laboratory animals, even well-trained pigs can become stressed when technicians take blood samples.
“In a typical study, the pigs would be snared, and technicians would take blood samples up to 24 times or more in a 24-hour study. The Culex-L approach is completely stress-free for them. They behave completely normally — they have freedom of movement, they can play, and pigs like to play. They don’t even seem to notice that the samples are being taken.”
Pigs are social animals and can become stressed when kept alone. “The U.S. Department of Agriculture has also used the system to look at environmental enrichment for sows that need to be isolated during birthing, to see what effect the availability of toys has on oxytocin levels, litter size, and immune response. The results from studies like these can then be used to advise farmers wanting to improve the yields from their pigs.”