Study into selected antimicrobial drugs for koalas (Phascolarctos cinereus)

235 Pages·2017·3.62 MB·English

by  Soraya Gharibi

Study into selected antimicrobial drugs for koalas
(Phascolarctos cinereus), incorporating consideration of
koalas’ endogenous plasma and serum antibacterial activity

Soraya Gharibi

A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy

Sydney School of Veterinary Sciences, Faculty of Science
The University of Sydney

September 2017
(Final version submitted March 2018)
Declaration
This thesis is submitted to The University of Sydney in fulfilment of the requirements for the
degree of Doctor of Philosophy.
The work presented in this thesis, to the best of my knowledge, is the product of my own work
with some contributions from other researchers. All the assistance received in preparing this
thesis and sources have been acknowledged.
I, hereby, declare that I have not submitted this material, in either full or in part, for a degree
at this or any other institution.
Soraya Gharibi
September 2017
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Acknowledgements
I would like to express my sincere gratitude to my supervisor, Associate Professor Merran
Govendir, for her great supervision during these three and half years. I could never have
fulfilled the candidature requirements without her unfailing support, guidance and invaluable
advice. She was always there to guide me, and I have learnt greatly from her extensive
comments and recommendations for writing this thesis. I want you to know how much I value
your excellent support with providing both general and scientific advice.
I also appreciate helpful advice of my co-supervisor, Dr Kong Li, during the HPLC method
development for the three drug assays.
I am sincerely grateful to my colleague, Dr Benjamin Kimble, who was always available
anytime to guide me in my project, even during weekends and out of hours. I have benefitted
greatly from his valuable guidance and assistance in technical and scientific aspects of my
project. I am very grateful to my colleague, Dr Aaron Izes, for his support and proofreading
many chapters of this research project, meticulously. Thanks to Dr Christie Budd for her
kindness and support in our laboratory during my candidature. Working with you was a great
honour and pleasure.
This thesis would not have been possible without the contribution of Dr Larry Vogelnest.
Although acknowledged later again, Dr Vogelnest was instrumental in obtaining most of the
koala samples and I am thankful for his involvement.
I am deeply thankful to Dr Peter Thomson who consented to proofread some chapters of my
thesis. I also appreciate his excellent advice and assistance during my candidature.
Many thanks to Judy Sung for the time she has taken to proofread a chapter of this thesis.
Many people have contributed to various aspects of this project. I would like to express my
heartfelt gratitude to all of them for their assistance in this project, in particular:
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To Dr Denise Wigney and Dr Angeles Sanchez-Perez (The University of Sydney) for their
advice on bioassays. Special thanks to Denise who was always willing to assist me in
conducting the bioassays and to provide me with the bacterial strains.
To Taronga Zoo and Los Angeles Zoo and their respective veterinarians, laboratory technical
officers, veterinary nurses and koala keepers for their participation in my project, particularly
Dr Larry Vogelnest, Paul Thompson, Dr Frances Hulst, Dr Kimberly Vinette Herrin, Dr
Gabrielle Tobias, Vanessa Di Giglio, Natalie Miller, Nicholas De Vos and Laura Jones at
Taronga Zoo for administering the drugs, collecting blood samples for the in-vivo studies, and
opportunistic blood samples, and providing accurate information about the koalas during this
project. I also benefited from the valuable perspective of Dr Vogelnest who edited the
manuscript of posaconazole paper, meticulously. Without the assistance of Paul Thompson,
the last chapter of this thesis would have not been completed during the candidature. Many
thanks to Drs Cindy Stadler at Los Angeles Zoo (CA, US) and Julie Barnes at Santa Barbara
Zoo (CA, US) who administered the posaconazole intravenously and collected the samples.
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. posaconazole administration.
To Dr Sabrina Lomax for providing blood samples of cows and sheep. Thank you Sabrina for
your kind cooperation and being so accurate in following the directions when collecting
samples for Chapter 7.
To Dr David Phalen and Dr Robin Bell for providing blood samples of possums and horses,
respectively.
To Christine Black for providing dog and cat plasma samples.
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To Cheyne Flanagan and Port Macquarie Koala Hospital, Dr Amber Gillett and Australia Zoo
Wildlife Hospital, Dr Fumie Tokonami and Currumbin Wildlife Hospital, Caroline Marschner
and Dr Iona Maher for providing blank koala samples for this project.
To Tess La-Lande with her lovely smile who was always kind and willing to assist me.
To Michelle Liu for her collaboration when developing the in-vitro cefovecin assay.
I thank all my office colleagues, particularly, Caroline Marschner, Kendy Tang, Judy Sung,
Mariko Yata, Kate Worthing, Dr Alan Marcus and Laura Schmertmann. I really appreciate all
your help and encouragement.
I am also deeply thankful to all the school staff in the McMaster building, Sydney School of
Veterinary Science for their generous support and providing facilities to complete the project,
especially Sally Pope, Veronica Ventura, George Tsoukalas and Christine Black.
Great thanks to the Sydney School of Veterinary Science that provided the Postgraduate
Student Scholarship in Veterinary Pharmacology for 3.5 years.
Great thanks to the Winifred Violet Scott Charitable Foundation and Sydney School of
Veterinary Science Whitehead Bequest that financially supported the cost of the consumables
for this project.
I would also like to express my heartfelt thanks to the examiners who spend their precious time
in reading this thesis.
I would like to acknowledge all the koalas and other animals that provided plasma samples for
this research. Our laboratory undertook this research to expand knowledge on Australian
marsupials and improve therapeutic outcomes for sick and injured koalas.
I am eternally grateful to my family and my friends. Special thanks to my father Ali and mother
Mastoureh for their support, protection and devotion. You have taught me to work hard for
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everything I am passionate for, to seek my dreams and never give up. I am thankful to my
lovely sisters Leila, Razieh, Narges, Sakineh and Hajar for their love and encouragement. To
my dear family: I sincerely appreciate everything you have done for me. Thank you for being
delighted with my achievements and for supporting me during the hard times.
Finally, my deepest appreciation to my husband Reza Taghipour for his unconditional love and
support. He spent a lot of time on editing the Figures of this thesis several times. I cannot thank
you enough for your encouragement and patience during those difficult days. You are unique.
Thank you for being caring and attentive.
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Publications and conference abstracts arising from this thesis
Journal articles
Gharibi, S., Kimble, B., Vogelnest, L., Barnes, J., Stadler, C.K. and Govendir, M., 2017.
Pharmacokinetics of posaconazole in koalas (Phascolarctos cinereus) after intravenous and
oral administration, Journal of Veterinary Pharmacology and Therapeutics, 40: 675-681.
Conference abstracts
1- Gharibi, S. and Govendir, M. Research update: Medicines for the koala (Phascolarctos
cinereus). 2nd National Koala Conference, 2-4 Jun 2017, Port Macquarie, NSW, Australia.
2- Gharibi, S., Kimble, B, Barnes, J., Stadler, C.K., Vogelnest, L. and Govendir, M.
Pharmacokinetics of posaconazole in koalas (Phascolarctos cinereus) after intravenous and
oral administration. Sydney School of Veterinary Science Conference, 9-10 Nov 2016, The
University of Sydney, NSW, Australia.
3- Gharibi, S., Kimble, B. and Govendir, M. In-vitro plasma protein binding of cefovecin in
the koala (Phascolarctos cinereus) vs the horse (Equus caballus). ANZCVS Science
Week, 7-9 Jul 2016, Gold Coast, Australia.
4- Gharibi, S., Kimble, B. and Govendir, M. In-vitro plasma protein binding of cefovecin in
some Australian marsupials and the horse. Sydney School of Veterinary Science Conference,
28-29 Oct 2015, The University of Sydney, NSW, Australia.
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Author contribution statement
Re the publication entitled:
Pharmacokinetics of posaconazole in koalas (Phascolarctos cinereus) after
intravenous and oral administration
I, Soraya Gharibi (PhD Candidate), was the chief investigator of the research documented in
this publication. I performed all the sample analyses for the p.o. study and undertook the data
analyses for both the p.o. and i.v. studies. I was the primary author of all drafts of the
manuscript and the chief respondent to the journal referees’ comments.
Dr Merran Govendir assisted the project as supervisor and finalising the manuscript for
publication. Dr Larry Vogelnest (Taronga Zoo, NSW) administered the posaconazole and
collected the blood samples at Taronga zoo. Dr Vogelnest reviewed all versions of the
manuscript. Drs Cindy Stadler and Julie Barnes administered the posaconazole and collected
the samples at Los Angeles Zoo (CA, US). Drs Stadler and Barnes reviewed all versions of the
manuscript. Dr Benjamin Kimble assisted the project on HPLC method development. Dr
Kimble reviewed all versions of the manuscript.
Soraya Gharibi 27/09/2017
I, as co-author, endorse that this level of contribution by myself and the candidate indicated
above is appropriate:
Merran Govendir 27/09/2017
Benjamin Kimble 27/09/2017
Larry Vogelnest 16/03/2017
Cindy Stadler 16/03/2017
Julie Barnes 19/03/2017

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Summary of the thesis
Studies on pharmacokinetic profiles on some first-line drugs for koalas argue that traditional
ad-hoc extrapolations of dosages from carnivorous species such as dogs and cats to the koala,
a folivore, are inappropriate. This research describes changes in plasma concentrations of
amoxicillin and cefovecin, currently administered by some veterinarians to koalas. A third
drug, posaconazole, was also investigated as its broad-spectrum antifungal activity might be
efficacious against cryptococcal infections in koalas. HPLC methods to determine plasma
concentrations of these antimicrobials were developed and validated. Posaconazole was
administered at 3 mg/kg to two koalas intravenously and 6 mg/kg to another six koalas orally.
Based on plasma concentrations, posaconazole is predicted to be efficacious for the treatment
of cryptococcosis in koalas. An in-vitro study to determine cefovecin binding to plasma
proteins of koalas and other selected Australian marsupials demonstrated the proportion of
binding between 12 to 40 %, suggesting that the elimination half-life of cefovecin in these
species is likely to be shorter than those in the dog and cat. Cefovecin was administered as a
single bolus (8 mg/kg) to six koalas subcutaneously. Cefovecin plasma concentrations at all
time points (0 to 96 h) in all animals were below 1 µg/mL, indicating cefovecin has a short
duration of action in koalas. Amoxicillin was administered to another six koalas at 10 mg/kg
subcutaneously. Low concentrations of amoxicillin were detected; however, drug instability
might have contributed towards these findings. Bioassays (agar disc diffusion and broth
microdilution inhibition assays) were undertaken to confirm amoxicillin and cefovecin HPLC
results. The bioassay demonstrated variable plasma antibacterial activities at t = 0 h (before
koalas were medicated). Consequently, the endogenous antibacterial activity of koala blood
matrices (plasma, serum, etc.) to inhibit E. coli and S. aureus were evaluated. Koala blood
matrices demonstrated significant variations in inhibiting both pathogens’ growth compared to
other species studied. Reasons for such variations were unclear but opened a new area for
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investigation into koalas’ endogenous antimicrobial activity and how it might be utilised to

assist this ‘vulnerable’ species control infectious diseases.
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