Part 1 published earlier this month described how colleague Benjamin Kimble developed an assay for measuring the concentration of tramadol and its active metabolite O-desmethyl tramadol in koala plasma. I also described how we determined the assay’s accuracy and validity.

This part, Part 2 describes using this assay to look at the rate of tramadol metabolism in the koala compared to that in the rat.

 Tramadol is a medicine registered for use in people. It is used for pain relief. It is very closely related to the opioid analgesics such as morphine and methadone. One of the advantages of tramadol is that it can be given by mouth to the patient.  Tramadol is used in veterinary practice. It is also being administered to koalas to provide pain relief to those animals that may have undergone trauma, such as being hit by cars or attacked by feral animals. The Koala Hospital at Port Macquarie, NSW, Australia http://www.koalahospital.org.au/ was interested in how effective tramadol is in koalas and generously provided some funds to develop the assay to detect the drug in the koala plasma.

You can imagine that if a wild koala has been hit by a car or attacked by a feral animal and it survives, that it has been highly stressed. It is hoped that injured wild koalas are found by people and are taken to wildlife hospitals for treatment.  However the new hospital environment and the handling of the animal further stresses them until they adapt to the surroundings, routines and people of the hospitals.  So there is a need to find an analgesic medicine for these animals that is effective and perhaps only needs to be administered every 24 h.  So one of our laboratory’s aims is to find an analgesic that is not only effective, but long lasting.

So for a medicine to be long lasting there are a few different strategies, however the first thing that needs to be checked out is how quickly the body eliminates the medicine.  The body has two main elimination processes:

  • Metabolism primarily occurs in the liver. The liver converts the parent medicine into another active medicine and / or inactive substances. These substances can then be excreted in the faeces and / or the urine. Tramadol is recognized to undergo metabolism in most species. In most species it is metabolized into one active metabolite called O-desmethyl tramadol and an inactive metabolite N-desmethyl tramadol.
  • The parent medicine is not chemically broken down but is eliminated by the kidneys and excreted in the urine.

 

How did Benjamin investigate the rate of tramadol metabolism?

We need a reference point to compare the rate of metabolism of tramadol in koalas so we compared it to the rate of tramadol metabolism in the rat. So we obtained metabolism enzymes (these enzymes are extracted from the liver and are called ‘microsomes’)

We purchase rat microsomes from a commercial supplier; we extract the koala microsomes from liver tissues. When a koala dies from being hit by a car, the wildlife hospitals collect a small amount of liver for these studies. No animal is killed for any of our studies.

So the same procedure is done with the rat microsomes and the koala microsomes. The tramadol is incubated with the microsomes, we then stop the reaction at set time points and look at the rate of disappearance of the tramadol and rate of appearance of the metabolites.

So if you look at the graphs provided. The black trace is the change in amplitude of the tramadol when incubated with rat microsomes. The tramadol peak is in the circle on the right (it appears at about 14 minutes after the sample goes through the machine) at the beginning of the incubation (top graph t = 0 min). The amplitude of the peak decreases at 30 minutes of incubation and the metabolites appears at in the orange circle after 30 minutes of incubation and then there is not much difference in the amplitude of the tramadol or metabolite after 60 minutes of incubation.

The pink trace is what is happening when the tramadol is incubated with the koala microsomes.  At the beginning of the assay the tramadol peak (in the blue circle) is the same amplitude as in the rat. But the tramadol peak does not decrease like it does in the rat at 30 and 60 minutes.

We still have a lot of work to do with this microsome assay to determine the rate of tramadol metabolism in koalas but what we have learned from this study is that koalas also metabolize tramadol to O-desmethyl tramadol and perhaps koalas slowly metabolize tramadol and thus it will have a long duration of analgesia in koalas.

Tramadol metabolism graphs

Some the readers will know that tramadol has side effects in people such as constipation; some of you will also know that some people metabolize tramadol quickly and some people metabolize tramadol slowly, so Benjamin and I know we still have a lot of work to do to investigate how effective tramadol may be in koalas.

 

Thanks for reading  🙂

Thanks to The Koala Hospital at Port Macquarie, NSW, Australia for funds to develop this assay.  

 Research projects available in my lab for 2018

Looking for people to undertake a research degree such as a research masters or Doctor of Philosophy (PhD) to take this research and many other projects, further. You will need the equivalent of a good undergraduate degree in biological sciences. For more information see my University of Sydney web page  http://sydney.edu.au/science/people/merran.govendir.php

or contact me directly via merran.govendir@sydney.edu.au