I am always lamenting how few people read our research, and the disconnection between the way we report our findings, versus the requirements for knowledge from people actually administering the treatments. We are nerdy scientists, and you kind people just trust that we know what we are talking about. However, we also have to prove to other scientists that our results are valid. The academic world (rightly so) requires us to publish our work, so that anyone, in any part of the world who understands pharmacokinetics can read our articles and say to themselves, “yep, if I had some koalas, and wanted to know about the PK of posaconazole in this species, I would probably do exactly the same things, or very similar, and probably get the same results”.
In this blog, I am attempting to translate essentially a whole academic journal article. Let us know what you think. If there is any interest, it may inspire me to provide translations of all the koala medicine papers.
Pharmacokinetics of posaconazole in koalas (Phascolarctos cinereus) after intravenous and oral administration S. Gharibi, B. Kimble, L. Vogelnest, J. Barnes, C. K. Stadler, M. Govendir
Posaconazole is an anti-fungal medication. Yeasts and fungi are everywhere in the environment, but occasionally koalas may develop active fungal infections with Cryptococcus neoformans and C. gattii. Infection usually starts in the nose and in severe cases can spread to the brain. Koalas have been treated with a variety of anti-fungal agents with mixed success. Recently, our research group (Black et al 2012 (Dr Lisa Black BVSc PhD) Govendir et al, 2016) reported that that neither fluconazole (10 mg /kg by mouth twice daily) nor a combination of itraconazole and amphotericin B, were likely to be effective treatments of these infections. These researchers demonstrated that the concentrations of these drugs did not reach levels in the bloodstream expected to kill the fungi. Posaconazole is a newer antifungal, which has demonstrated good efficacy in treating similar fungal infections in people.
This study was conducted at two sites using healthy, non-infected koalas held permanently at zoos, i.e. LA Zoo and Taronga Zoo. 2 senior female koalas were administered a single dose of posaconazole intravenously. Six koalas of mixed sex and ages were administered posaconazole suspension by mouth. It is a long story why some of the study was done in the US. However at the time the injectable formation was only available in the US, so Los Angeles zoo decided to help and look at the pharmacokinetic profile in two koalas when injected into the vein.
All the koalas in the study were briefly anaesthetised and a catheter placed in a vein in their forearm to enable blood collection. The koalas at LA Zoo (Killarney and Georgie) were given their medication into the vein while under anaesthesia. The koalas at Taronga took their medication by syringing it into their mouth, and did not put up much fuss, so it must not have tasted too bad for them. As usual for a pharmacokinetic study, blood samples were taken from the catheter, at multiple time points after treatment. All of the koalas were healthy after treatment, during blood sampling and no adverse effects were noted in the following weeks.
The blood samples were centrifuged and the top plasma layer was stored frozen prior to analysis. The Veterinary Pharmacology Laboratory at the University of Sydney has two high-performance liquid chromatography machines, which we refer to as “Fred” and “George” to distinguish them. Fred, who has the UV-detector, was used for the analyses performed in Australia. The samples from the LA Zoo koalas were analysed at the Clinical Pharmacology Laboratory at the College of Veterinary Medicine at North Carolina State University.
Known quantities of posaconazole were added to blank (unmedicated) koala plasma to construct calibration curves, which allowed quantification of drug concentrations in the experimental samples. The “unknown” samples were analysed three times, on separate days. The drug assay had been shown to have a precision greater than 95% and an accuracy of greater than 90%. The amount of posaconazole bound to blood proteins in koalas was also measured.
Drug concentration versus time curves was plotted for both oral and intravenous routes, which allowed the mathematics of pharmacokinetic analysis to be performed. Amongst other things, the half life, average maximal drug concentration, the time at which the maximum drug concentration occurred, the rate at which posaconazole was cleared from the body, the amount of time drug was present in the bloodstream, and the amount of drug taken into the blood after being swallowed, were calculated.
Posaconazole vs time intravenous Posaonazole vs time oral
As good scientists, we always like to point out the flaws in our own work.
Results / Discussion
Posaconazole was highly bound to plasma proteins (99%). Following intravenous administration to koalas, posaconazole half-life was shorter than that in dogs, cats and people, and it was cleared by koalas faster than cats or dogs. When given orally, posaconazole has a longer half-life than fluconazole. Interestingly, the highest drug concentration after oral treatment was higher than that achieved in dogs, and more than 50 % of the drug administered found its way into the bloodstream.
This is the first time that good drug absorption has been documented in an orally administered treatment of koalas. The combination of good drug uptake, koalas taking the medicine well, it not appearing to cause side effects and having the potential to work, makes this a drug worth considering for koalas. The big question, “Will posaconazole be a useful treatment of fungal infections in koalas?” Well, for the first time, we are saying, yes, maybe.
Unfortunately, we don’t know exactly what drug concentrations will kill the fungal organisms we are interested in (Cryptococcus gattii). One indicator of what constitutes an effective treatment for these types of infection is AUC24/MIC (which roughly means, how much greater is the amount of drug in the body over a whole day, that that required to limit growth of the organism). For similar organisms an AUC24/MIC of 25 or greater is aimed for. Following the dosages administered to koalas in this study (both intravenously and orally) an AUC24/MIC of 38 was calculated- yippee!
So, at least in theory, we seem to have a drug that koalas appear to tolerate, is easily administered, can be given once a day and has a real hope of killing the infectious agents. There are still plenty of reasons why it might not work, but on paper (in the lab), we have a treatment that looks promising. Of course there has to be a downside (or seven)- this is wildlife medicine after all. In Australia, the drug costs about $700/ bottle.
This is a preliminary study, with few animals; further work would be required to investigate this treatment in larger numbers of patients and to optimise the dosage. It would also need to be trialled to see if it actually works for sick koalas.
How did this study and paper come about? Soraya Gharibi did most of the laboratory work and worked on it as part of her PhD. Soraya validated and performed the assays in Australia.
Benjamin helped with modifying the HPLC conditions for the assay.
Larry oversaw and performed treatments and blood sampling on the Australia koalas.
Cindy Stadler and Julie Barnes oversaw and performed treatments and blood sampling on the USA koalas.
Merran instigated the study and participated in obtaining the samples from the LA Zoo while on sabbatical at the College of Veterinary medicine, North Carolina State University (2014).
My name is Christie; I am a former student of Merran’s. I had no involvement in this study and am just the translator (any mistakes in the way this work has been presented are mine alone).
For the nerds, please read the full paper in Veterinary Pharmacology and Therapeutics 2017.
This study was financially supported by the Winifred Violet Scott Foundation. The authors appreciate the participation of the Taronga Zoo and Los Angeles Zoo and their respective veterinarians, veterinary nurses and koala keepers. Thanks to Paul Thompson at Taronga Zoo for preparation and shipping of samples. Great thanks to Professor Mark G. Papich and the Clinical Pharmacology Laboratory at the College of Veterinary Medicine, North Carolina State University, NC, USA, for assaying the plasma samples resulting from i.v. administration. Thanks to Dr Geoff Pye for his introduction to Los Angeles Zoo.