Evaluation of firocoxib inhibition of cyclooxygenase activity in alpacas by Abigail Chadwick A ...

53 Pages·2017·1.13 MB·English

by  Abi Chadwick

Evaluation of firocoxib inhibition of cyclooxygenase activity in alpacas

by
Abigail Chadwick

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of
the requirements for the
degree of

Honors Baccalaureate of Science in Animal Sciences
(Honors Associate)

Presented May 26, 2017
Commencement June 2017

AN ABSTRACT OF THE THESIS OF

Abigail Chadwick for the degree of Honors Baccalaureate of Science in Animal Sciences presented
on May 26, 2017. Title: Evaluation of firocoxib inhibition of cyclooxygenase activity in alpacas.

Abstract approved:_____________________________________________________
Katja F. Duesterdieck-Zellmer

Non-steroidal anti-inflammatory drugs (NSAIDs) are used to control pain and
inflammation, but can have serious adverse effects, including gastrointestinal ulceration. Non-
selective NSAIDs inhibit both cyclooxygenase isoforms: COX-1 and COX-2. COX-2 is
primarily responsible for the inflammatory response, while COX-1 is constitutively expressed,
representing the dominant source of prostaglandins for gastric epithelial cytoprotection. The
coxib class of drugs selectively reduces the activity of COX-2 while sparing protective effects of
COX-1 activity. Our goal is to determine a safe and therapeutic dose of firocoxib in alpacas.
There is no established method for measuring inhibition of COX-1 and COX-2 activity in
alpacas. It was our aim to validate quantification of clot-induced production of thromboxane B
2
(TXB ) as a measure for COX-1 activity, and lipopolysaccharide-induced prostaglandin E
2 2
(PGE )to assess COX-2 activity and to determine how firocoxib impacts COX activity.
2
Concentrations of TXB or its metabolite, 11-dehydrothromboxane B were not different
2 2
between alpaca plasma and serum with or without acetone or methanol purification, indicating
inability of the utilized ELISA to quantitate TXB or its metabolite in alpacas. PGE increased
2 2
with lipopolysaccharide exposure and 100 pg/ml firocoxib inhibited PGE production by at least
2
80% in all animals tested, demonstrating firocoxib’s therapeutic potential in alpacas.

Key Words: alpaca, firocoxib, NSAID, cyclooxygenase, camelid, prostaglandin, thromboxane

Corresponding e-mail address: [email protected]
©Copyright by Abigail Chadwick
May 26, 2017
All Rights Reserved
Evaluation of firocoxib inhibition of cyclooxygenase activity in alpacas

by
Abigail Chadwick

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of
the requirements for the
degree of

Honors Baccalaureate of Science in Animal Sciences
(Honors Associate)

Presented May 26, 2017
Commencement June 2017
Honors Baccalaureate of Science in Animal Sciences project of Abigail Chadwick presented on
May 26, 2017.

APPROVED:

_____________________________________________________________________
Katja F. Duesterdieck-Zellmer, Mentor, representing Department of Clinical Sciences

_____________________________________________________________________
Maureen Larson, Committee Member, representing Department of Clinical Sciences

_____________________________________________________________________
Stacy Semevolos, Committee Member, representing Department of Clinical Sciences

_____________________________________________________________________
Toni Doolen, Dean, Oregon State University Honors College

I understand that my project will become part of the permanent collection of Oregon State
University, Honors College. My signature below authorizes release of my project to any reader
upon request.

_____________________________________________________________________
Abigail Chadwick, Author
Acknowledgements

I would like to thank my mentor, Dr. Zellmer, for encouraging me to pursue research in
an area of the veterinary field that I was unfamiliar with at the time we met. She has taught me so
much about academic writing, jugular venipuncture, and everything in between. I simply would
not have and could not have done this research without her.
I would also like to thank Maureen Larson, who not only showed me the ropes of all the
bench-work and helped me trouble-shoot all the issues that came up with the ELISAs, but she
became an important friend and moral support for me in hard times.
Thank you to Dr. Semevolos for her guidance and support as a valuable committee
member.
Thank you to Dr. Klopfenstein and Dr. Vanegas in Rural Veterinary practice for helping me with
exams on our alpacas and with sample collection. Thank you to Kasey Pedder, Peter McPartlin,
and the rest of the Large Animal Hospital Husbandry Team for their assistance caring for and
restraining animals used for sample collection.
Thank you to the OSU Department of Clinical Sciences for funding for this research.
Table of Contents
Introduction ...................................................................................................................................... 1
Literature Review............................................................................................................................. 2
Objectives and Aims ........................................................................................................................ 8
Materials and Methods ..................................................................................................................... 9
Sample Collection ........................................................................................................................ 9
Determination of COX-1 Activity ............................................................................................... 9
Experiment 1 – Initial Quantitation of Thromboxane B in Alpaca Blood ............................ 10
2
Experiment 2 – Quantitation of TXB inAlpaca Blood at Lower Dilutions and the Use of
2
Equine Blood as a Positive Control ....................................................................................... 11
Experiment 3 – Quantitation of Thromboxane B Using Different Purification Procedures in
2
Alpaca and Equine Serum and Plasma .................................................................................. 11
Experiment 4 – Quantification of 11-dehydrothromboxane B in Alpaca Serum and Plasma,
2
Using different Purification Procedures ................................................................................. 13
Determination of COX-2 Activity ............................................................................................. 14
Experiment 5 – Initial Quantitation of Prostaglandin E in Alpaca Blood exposed to LPS ...... 14
2
COX-2 Inhibition by Firocoxib ................................................................................................. 16
Experiment 6 – Preliminary Experiment to Determine an Appropriate Range of Firocoxib
Concentrations ....................................................................................................................... 16
Experiment 7 –Determination of COX-2 Inhibition by Firocoxib in 3 Alpacas .................... 18
Data Analysis ......................................................................................................................... 18
Results ............................................................................................................................................ 19
Sample Collection ...................................................................................................................... 19
Determination of COX-1 Activity ............................................................................................. 19
Experiment 1 – Initial Quantitation of Thromboxane B in Alpaca Blood ............................ 19
2
Experiment 2 – Quantitation of TXB inAlpaca Blood at Lower Dilutions and the Use of
2
Equine Blood as a Positive Control ....................................................................................... 22
Experiment 3 – Quantitation of Thromboxane B Using Different Purification Procedures in
2
Alpaca and Equine Serum and Plasma .................................................................................. 23
Experiment 4 – Quantification of 11-dehydrothromboxane B in Alpaca Serum and Plasma,
2
Using different Purification Procedures ................................................................................. 25
Determination of COX-2 Activity ............................................................................................. 26
Experiment 5 – Initial Quantitation of Prostaglandin E in Alpaca Blood exposed to LPS ... 26
2
COX-2 Inhibition by Firocoxib ................................................................................................. 27
Experiment 6 – Preliminary Experiment to Determine an Appropriate Range of Firocoxib
Concentrations ....................................................................................................................... 27
Experiment 7 – Determination of COX-2 Inhibition by Firocoxib in 3 Alpacas ................... 28
Discussion ...................................................................................................................................... 32
Conclusions .................................................................................................................................... 37
Literature Cited .............................................................................................................................. 38
Abbreviations ................................................................................................................................. 42
Table of Figures
Figure 1 – Biosynthesis of prostanoids via cyclooxygenase ........................................................... 3
Figure 2 – Comparing structure of COX isoforms18 ....................................................................... 4
Figure 3 – Structure of firocoxib ..................................................................................................... 5
Figure 4 – Clot-induced TXB concentrations measured using a commercially available ELISA
2
(Thromboxane B ELISA Kit, Cayman Chemical, # 501020) in alpaca serum (1 and 2-hour
2
clotting times) and plasma (as negative control) at higher dilutions, without purification. Data
represents pooled plasma or serum from two animals. .................................................................. 21
Figure 5 – Clot-induced TXB concentrations measured using a commercially available ELISA
2
(Thromboxane B ELISA Kit, Cayman Chemical, # 501020) in alpaca serum (1 and 2-hour
2
clotting times) and plasma (as negative control) at higher dilution, with methanol purification.
Data represents pooled plasma or serum from two animals. ......................................................... 21
Figure 6 – Clot-induced TXB concentrations measured using a commercially available ELISA
2
(Thromboxane B ELISA Kit, Cayman Chemical, # 501020) in alpaca serum (1 and 2-hour
2
clotting times) and plasma (as negative control) at lower dilutions, without purification. Data
represents pooled plasma or serum from two animals. .................................................................. 22
Figure 7 – Clot-induced TXB concentrations measured using a commercially available ELISA
2
(Thromboxane B ELISA Kit, Cayman Chemical, # 501020) in equine serum (1 and 2-hour
2
clotting times) and plasma (as negative control) at various dilutions, without purification. These
samples were used to ascertain adequate performance of the ELISA in our laboratory. Data
represents measurements in blood from n=1 horse. ....................................................................... 23
Figure 8 – Clot-induced TXB concentration in horse serum (allowed to clot for 1 hour) and
2
plasma (as negative control), measured with a commercially available ELISA (Thromboxane B
2
ELISA Kit, Cayman Chemical, # 501020) from 1 animal, using different purification methods. 24
Figure 9 – Clot-induced thromboxane B concentration in alpaca serum (allowed to clot for 1
2
hour) and plasma (as negative control), determined with a commercially available ELISA
(Thromboxane B ELISA Kit, Cayman Chemical, # 501020) from pooled blood of 2 animals,
2
using different purification procedures. ......................................................................................... 24
Figure 10 – Clot-induced 11-dehydroTXB concentration in alpaca serum and plasma (as
2
negative control), determined with a commercially available ELISA (11-dehydro Thromboxane
B ELISA Kit – Monoclonal, Cayman Chemical, # 519510) from 1 animal using different
2
purification procedures. ................................................................................................................. 25
Figure 11 – PGE concentrations in alpaca plasma as measured with a commercially available
2
ELISA (Prostaglandin E ELISA Kit - Monoclonal, Cayman Chemical, # 514010) from pooled
2
blood of 2 animals. ......................................................................................................................... 26
Figure 12 – Percent Inhibition of COX-2 activity in alpaca plasma as measured with a
commercially available ELISA (Prostaglandin E ELISA Kit - Monoclonal, Cayman Chemical, #
2
514010) from pooled plasma of 2 animals. Percent inhibition of COX-2 activity was calculated
by the following formula: % 𝐶𝑂𝑋−2 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝑃𝐺𝐸2𝑐𝑜𝑛𝑡𝑟𝑜𝑙−
𝑃𝐺𝐸2𝑓𝑖𝑟𝑜𝑐𝑜𝑥𝑖𝑏𝑃𝐺𝐸2𝑐𝑜𝑛𝑡𝑟𝑜𝑙×100 with the blue dots representing a dataset calculated using
the LPS conditioned samples that had the treatment vehicle added (DMSO) as control sample,
and with the orange dots representing a dataset calculated using the LPS conditioned samples that
did not have the treatment vehicle added as control sample. ......................................................... 28
Figure 13 – COX-2 Inhibition by Firocoxib calculated using LPS untreated control. Data
represents individual measurements in blood from 3 alpacas. The blue dotted curve represents the