AN ABSTRACT OF THE DISSERTATION OF Sultan M. Alshahrani for the degree of Doctor of Philosophy in Pharmacy presented on November 22, 2017 Title: Pharmacokinetics of Flunixin in Elephants, Cefovecin in African Lions, and Gamithromycin in Alpacas Abstract approved: J Mark Christensen Foot pathology is a real problem in captive elephants. Osteoarthritis is the major concern among musculoskeletal disorders and foot health problems for elephants in North America zoos with one- half of Elephants in captivity showing foot health care problems in zoos. A higher ratio of captive Asian elephants (Elephas maximus) is affected by osteoarthritis than African Elephants according to Association of Zoos and Aquarium (AZA). The incidence of arthritis increases as the age of the elephant s increases that often requires therapeutic drug monitoring. Flunixin is the one of most commonly prescribed NSAID agent in elephants. Flunixin is a potent non- steroidal anti- inflammatory agent (NSAID)
that inhibits cyclooxygenase enzymes (COX). Flunixin concentration was successfully determined in Asian and African elephants plasma using a developed HPLC- UV method that was validated according to U.S Food and Drug Administration (FDA) guidelines. The pharmacokinetics of flunixin was evaluated in both elephant species after administration of low and high doses (0.8 mg/kg and 1.5 mg/kg) using non- compartmental analysis. The flunixin pharmacokinetics showed no significant statistical differences between two elephant species and the maintenance flunixin oral dose, which can manage osteoarthritis in elephants, may be 0.8 mg/kg of body weight. The pharmacokinetic of cefovecin (macrolide- like antibiotic) was evaluated in African lions (Panthera leo) after administration of low and high doses (4 and 8mg/kg) subcutaneously. Cefovecin plasma conacentrations were successfully determined using an HPLC- UV methad that was developed and validated based on FDA bioanalytical requirements. The pharmacokinetics of cefovecin showed that allometric extrapolation (based on animal s weight) of the cefovecin dose from smaller animals (cats, and dogs) to larger animals (lions) was not appropriate.
The pharmacokinetics of gamithromycin (long acting antibiotic) was evaluated in Alpaca Plasma, Lung Bronchoalveolar Lavage Cells, Pulmonary Epithelial Lining Fluid, and Plasma Polymorphonuclear Cells after subcutaneous administration. The results indicated that gamithromycin has long elimination half- life in plasma and distributed rapidly into pulmonary components. Gamithromycin remains above or near the minimum bacterial inhibitory concentration at the site of action (lung tissues) for a week.
Copyright by Sultan M. Alshahrani November 22, 2017 All Rights Reserved
Pharmacokinetics of Flunixin in Elephants, Cefovecin in African Lions, and Gamithromycin in Alpacas By Sultan M. Alshahrani A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented on November 22, 2017. Commencements June 2018
Doctor of Philosophy dissertation of Sultan M. Alshahrani presented on November 22, 2017. APPROVED: Major Professor, representing Pharmacy Dean of the College of Pharmacy Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Sultan M. Alshahrani, Author
ACKNOWLEDGEMENTS It gives me pleasure to express my sincere and utmost acknowledgment with gratitude to my major advisor Prof. JM Christensen for his extraordinary support and guidance. I consider myself privileged after he shared his extraordinary experience and science with my colleagues and me. He inspired me with many aspects inside and outside education life. I am highly appreciated to my graduate committee members Dr. Adam Alani, Dr. Oleh Taratula, and Dr. Conroy Sun for their guidance, help, and encouragement during my study. I would like to acknowledge Dr. Deidre Johns of Veterinary Medicine School, Oregon State University, for sharing her valuable time to act as a graduate school representative on my committee. I thank all of College of Pharmacy staff and faculties especially Mrs. Debra Peters, administration assistant at College of Pharmacy, for her help and assistance during my study. I am thankful for the King Khalid University and the Saudi Arabian Culture Mission for giving me a golden chance to complete my higher education and for their help and assistance during my stay in the United States.
I am highly appreciated for the help and support that were given by my brothers and sisters. Also, I would extend thanks to my friends Abdulah Alanazi, Adel Alfatease, Hassan Albarqi, Ali Alsalhi, Wisam Selman, Sultan Alanzi, and Fawaz Alazmi for their support and help throughout my stay in the United States. I am forever grateful for my Mother Jamla Mohammed Zewifer, for her support, thoughts, and prayers overseas, and without all of that, I wouldn't be who I am. Without the significant support, sacrifices, and love of my wife Jawharah Nasser, I wouldn't be able to achieve success. I am truly fortunate to have such a great woman in my life.
TABLE OF CONTENTS Page Flunixin determination in elephant s plasma method development and the pharmacokinetics of flunixin after intravenous and oral administration to African and Asian elephants (Loxodonta africana and Elephas maximus). 1 Chapter 1: General introduction. 1 1- Literature review... 2 2- Flunixin meglumine. 6 References..... 9 Chapter 2: High performance liquid chromatography method development and validation for the determination of flunixin in Asian elephants plasma 17 Abstract. 17 Introduction. 18 Materials and Methods.. 21 Reagents and Chemicals.... 21 Instrumentation... 21 Method development and designs.... 21 Chromatographic conditions...... 22 Standard solutions and calibration curves preparation 23 Sample preparation......... 24 Results.. 25 Appropriateness of chromatographic conditions.... 25 Method validation..... 25
TABLE OF CONTENTS (Continued) Page Linearity. 30 Limit of quantification (LOQ) and Limit of detection (LOD). 33 Accuracy... 33 Precision.. 33 Selectivity 36 Robustness..... 36 Application.... 37 Discussion.... 40 Conclusion... 42 References......... 43 Chapter 3: Pharmacokinetics of flunixin after intravenous and oral administration to African and Asian elephants (Loxodonta africana and Elephus maximus).. 48 Abstract...... 48 Introduction... 50 Determination of flunixin in elephant s plasma.. 53 Materials and Methods.... 53 Flunixin Analysis. 53 Reagents and Chemicals.... 53 Instrumentation...... 53 Method development and design.. 54 Standard solutions and calibration curves preparation... 54
TABLE OF CONTENTS (Continued) Page Sample preparation....... 55 Study design and animals.. 56 Sample collection.. 57 Pharmacokinetic and statistical analyses. 57 In Vitro plasma- protein binding study.. 58 Results.... 61 Pharmacokinetics of flunixin.. 61 In Vitro protein binding... 74 Discussion....... 75 Conclusion.. 79 References...... 80 Chapter 4: Determination of cefovecin in African lion s plasma with HPLC- UV: method development development and validation..... 87 Abstract...... 87 Introduction... 88 Materials and Methods.... 90 Reagents and Chemicals.. 90 Instrumentation...... 90 Method development and chromatographic conditions... 91 Standard solutions and calibration curves preparation... 91 Sample preparation....... 92
TABLE OF CONTENTS (Continued) Page Results.. 93 Sample preparation and plasma analysis....... 93 Method validation criteria.... 98 Selectivity...... 98 Linearity....... 100 Limit of detection (LLOD) and Limit of quantification (LLOQ). 100 Accuracy........ 104 Precision.... 104 Discussion.... 106 Conclusion...... 110 References..... 111 Chapter 5: The pharmacokinetics of cefovecin in African lions (Panthera leo) foolowing subcutaneous administration....... 114 Abstract...... 114 Introduction.. 115 Plasma Analysis....... 118 Materials and Methods..... 118 Reagents and Chemicals.. 118 Instrumentation...... 118 Method development and chromatographic conditions. 119 Sample preparation.... 119 Pharmacokinetic study design.. 120
TABLE OF CONTENTS (Continued) Page Results... 121 Discussion.. 129 Conclusion...... 132 References.... 133 Chapter 6: Pharmacokinetics of gamithromycin in alpaca plasma, lung bronchoalveolar lavage cells, pulmonary epithelial lining fluid, and plasma polymorphonuclear cells....... 136 Abstract...... 136 Introduction... 137 Materials and Methods.. 139 Gamithromycin formulation.. 139 Design and treatment. 140 Sample collection and gamithromycin analysis... 140 Calculation of gamithromycin concentrations in BAL Cells and PELF 142 Pharmacokinetic analysis... 142 Results.... 143 Discussion... 154 Conclusion...... 157 References.... 158 Chapter 7: GENERAL CONCLUSIONS... 162 Bibliography. 165 Appendices 184
LIST OF FIGURES Figure: Page Flunixin determination in elephant s plasma method development and the pharmacokinetics of flunixin after intravenous and oral administration to African and Asian elephants (Loxodonta Africana and Elephas maximus).... 1 Chapter 1: General introduction. 1 Figure 1: Flunixin metabolism in the liver.... 7 Chapter 2: High performance liquid chromatography method development and validation for the determination of flunixin in Asian elephants plasma.. 17 Figure 1: Chemical structure of flunixin. 18 Figure 2: Method II (50:20:30) elephant plasma spiked with 10 μg/ml flunixin and internal standard (Diclofenac Na + ) 15μL injection volume... 26 Figure 3: Method I (50:50) elephant plasma spiked with 10 μg/ml flunixin and internal standard (Diclofenac Na + ) 15μL injection volume.... 27 Figure 4: Method I (50:50) Blank elephant plasma spiked with internal standard (Diclofenac Na + ) 15μL injection volume....... 28 Figure 5: Method II (50:30:20) Blank elephant plasma spiked with internal standard (Diclofenac Na + ) 15μL injection volume.... 29 Figure 6: Calibration curve of Method I ACN: Water at ratio of 50:50 31 Figure 7: Calibration curve of Method I ACN:Methanol:Water at ratio of 50:20:30.... 32 Figure 8: Flunixin plasma concentration versus Time curve in Asian elephant after 0.8 mg/kg oral dose.... 38
LIST OF FIGURES (Continued) Figure Page Chapter 3: Pharmacokinetics of flunixin after intravenous and oral administration to African and Asian elephants (Loxodonta africana and Elephus maximus).. 48 Figure 1: Average flunixin plasma concentration versus time curve after administration of low dose (0.8mg/kg) orally to Asian elephant (Log- scale)... 62 Figure 2: Average flunixin plasma concentration versus time curve after administration of low dose (0.8mg/kg) orally to African elephant (Log- scale)... 63 Figure 3: Average flunixin plasma concentration versus time curve after administration of high dose (1.5 mg/kg) orally to Asian elephant (Log- scale)... 64 Figure 4: Average flunixin plasma concentration versus time curve after administration of high dose (1.5 mg/kg) orally to African elephant (Log- scale)... 65 Figure 5: Average flunixin plasma concentration versus time curve after administration of low dose (0.8mg/kg) intravenously to Asian elephant (Log- scale)... 66 Chapter 4: Determination of cefovecin in African lion s plasma with HPLC- UV: method development development and validation..... 87 Figure 1: Chemical structure of cefovecin sodium.... 88 Figure 2A: Blank lion plasma chromatogram..... 94 Figure 2B: Cefovecin chromatogram at 1 μg/ml concentration in lion plasma.... 95 Figure 2C: Cefovecin chromatogram at 10 μg/ml concentration in lion plasma.... 96 Figure 2D: Cefovecin chromatogram at 100 μg/ml concentration in lion plasma.... 97 Figure 3A: Cefovecin chromatogram at 100 μg/ml concentration in methanol (selectivity). 98
Figure LIST OF FIGURES (Continued) Page Figure 3B: Cefovecin chromatogram at 100 μg/ml concentration in lion plasma (selectivity)... 99 Figure 4: Concentration (μg/ml ) versus UV- Absorbance peaks average calibration curve of cefovecin levels range (0.1 100 μg/ml ).. 101 Figure 5: Residual curve that shows cefovecin concentration (μg/ml ) versus UV- absorbance AUC regression line model residuals over six runs for four days... 102 Figure 6: Studentized residual curve that shows cefovecin concentration (μg/ml) versus UV- absorbance AUC regression line model residuals over six runs for four days... 103 Figure 7: Cefovecin plasma concentration versus Time curve in African lion after 4 mg/kg subcutaneous dose.. 108 Chapter 5: The pharmacokinetics of cefovecin in African lions (Panthera leo) following subcutaneous administration....... 114 Figure 1: Cefovecin plasma concentration versus Time curve for all animals (n=6) after subcutaneous dose of 4mg/kg (low dose).. 123 Figure 2: Cefovecin plasma concentration versus Time curve for all animals (n=6) after subcutaneous dose of 8mg/kg (high dose).... 124 Figure 3: Average cefovecin plasma concentration versus Time curve after subcutaneous administration of 4mg/kg (n=6).. 125 Figure 4: Average cefovecin plasma concentration versus Time curve after subcutaneous administration of 8mg/kg (n=6).. 126
Figure LIST OF FIGURES (Continued) Page Chapter 6: Pharmacokinetics of gamithromycin in alpaca plasma, lung bronchoalveolar lavage cells, pulmonary epithelial lining fluid, and plasma polymorphonuclear cells.......... 136 Figure 1: Chemical structure of gamithromycin..... 140 Figure 2: Gamithromycin plasma concentration versus time curves after subcutaneous administration to six alpacas. 145 Figure 3: Average gamithromycin plasma concentration versus time curve after subcutaneous administration to six alpacas. 146 Figure 4: Gamithromycin concentration versus time curves in PELF after subcutaneous administration to four alpacas. 147 Figure 5: Average gamithromycin concentration versus time curve in PELF after subcutaneous administration to four alpacas 148 Figure 6: Gamithromycin concentration versus time curves in BAL cells after subcutaneous administration to four alpacas.. 149 Figure 7: Average gamithromycin concentration versus time curve in BAL cells after subcutaneous administration to four alpacas 150 Figure 8: Gamithromycin concentration versus time curves in PMNs after subcutaneous administration to four alpacas.. 151 Figure 9: Average gamithromycin concentration versus time curve in PMNs after subcutaneous administration to four alpacas 152 Figure 10: Point- to- point gamithromycin concentration comparison curve between plasma, BAL cells, PELF, and PMNs in alpaca 155
Table LIST OF TABLES Page Chapter 2: High performance liquid chromatography method development and validation for the determination of flunixin in Asian elephants plasma... 17 Table 1: Method I Accuracy and Precision.. 34 Table 2: Method II Accuracy and Precision. 35 Table 3: Pharmacokinetic parameters for an Asian elephant using the proposed HPLC- UV method (Method II) after administration of flunixin as 0.8 mg/kg (of body weight) orally.... 39 Chapter 3: Pharmacokinetics of flunixin after intravenous and oral administration to African and Asian elephants (Loxodonta africana and Elephus maximus)..... 48 Table 1: Asian elephants PK Parameters of flunixin after administration of 0.8mg/kg dose (low dose) orally 68 Table 2: African elephants PK Parameters of flunixin after administration of 0.8mg/kg dose (low dose) orally 69 Table 3: Asian elephants PK Parameters of flunixin after administration of 1.5 mg/kg dose (high dose) orally..... 70 Table 4: African elephants PK Parameters of flunixin after administration of 1.5 mg/kg dose (high dose) orally..... 71 Table 5: Asian elephants PK Parameters of flunixin after intravenous Administration of 0.8 mg/kg dose.... 72
Table LIST OF TABLES (Continued) Page Chapter 4: Determination of cefovecin in African lion s plasma with HPLC- UV: method development development and validation..... 87 Table 1: Inter- and Intraday HPLC- UV method Accuracy and Precision..... 105 Table 2: Pharmacokinetic parameters for an African lion using the proposed HPLC- UV method after single subcutaneous administration of cefovecin... 109 Chapter 5: The pharmacokinetics of cefovecin in African lions (Panthera leo) following subcutaneous administration....... 114 Table 1: African lion pharmacokinetic parameters of cefovecin after administration of 4 mg/kg dose (low dose) subcutaneously.... 127 Table 2: African lion pharmacokinetic parameters of cefovecin after administration of 8 mg/kg dose (high dose) subcutaneously.. 128 Chapter 6: Pharmacokinetics of gamithromycin in alpaca plasma, lung bronchoalveolar lavage cells, pulmonary epithelial lining fluid, and plasma polymorphonuclear cells....... 136 Table. 1: Pharmacokinetic parameters of gamithromycin in plasma, BAL cells, PMN, and PELF after subcutaneous administration of 6 mg/kg dose in 4 and 6 animals..... 153
1 Flunixin Determination in Elephant's Plasma Method Development and the Pharmacokinetics of Flunixin after Intravenous and Oral Administration to African and Asian Elephants (LOXODONTA AFRICANA and ELEPHAS MAXIMUS). SULTAN M. ALSHAHRANI 1,2, URSULA BECHERT 3, JM CHRISTENSEN 1* 1- Pharmaceutical Science Department, College of Pharmacy, Oregon State University, Corvallis, OR 2- Clinical Pharmacy Department, College of Pharmacy, King Khalid University, Abha, Saudi Arabia 3- College of Liberal and Professional Studies, University of Pennsylvania, Philadelphia, PA Chapter 1: General Introduction Asian elephants (Elephas maximus) are endangered according to the International Union for Conservation of Nature (IUCN) Red List of threatened species [1]. Approximately one- third of Asian elephants on the planet live in captivity in various countries, and another 1000 elephants live in zoos around the world [2,3]. On the other hand, African elephants (Loxodonta africana) were listed in Appendix I of the convention on international trade in endangered species (CITES). There are about 1,000 African elephants that live in captivity and zoos around the world [4]. Osteoarthritis is one of the major concerns that may lead to perilous consequences in an elephant's life. Non- Steroidal anti- inflammatory drugs have the upper hand in
2 the treatment of osteoarthritis in elephants. Flunixin (potent NSAID) has been widely used in veterinary medicine as anti- inflammatory and analgesic agent [5-7]. The overall purpose of this project was to determine flunixin pharmacokinetics in elephants and then to provide a proper dosage regimen to manage osteoarthritis in Asian and African elephants. To accomplish these objectives, a High Performance Liquid Chromatography method was first developed and validated to evaluate flunixin concentration in elephant's plasma. Then a flunixin pharmacokinetic study was performed to determine its absorption, distribution, and elimination after intravenous and oral administration. 1. Literature review: The adverse effect of captivity on elephant's foot health is strikingly high. Foot pathology is a real problem in captive elephants. Osteoarthritis is the primary concern among musculoskeletal disorders and foot health problems in North American zoos. One- half of Elephants in captivity exhibited foot health care problems across 63 zoos according to medical records. Asian elephants (Elephas maximus) are reported to have a higher ratio of captive elephants that are affected by osteoarthritis according to Association of Zoos and Aquarium (AZA). Arthritis incidence increases as the age of elephant's increases [8-10]. As captivity decreases the elephant's mobility, this environment generates an excessive increase in pressure being applied to the joints and synovial fluids. Furthermore, captive elephants suffer from an abnormal weight gain resulting in an
3 overload on leg bones and feet. As a result, bones undergo deformity and degeneration to compensate for the extra compression exerted by increased weight, and a low blood supply to leg tissues ensues. These types of extreme conditions affect the synovial fluid flow and collagen regeneration and leads to cartilage erosion and chondrocyte necrosis [14,15]. Many other factors are affected by arthritis in elephants such as an increase in Mycoplasma antibodies titre, and rising of rheumatoid factor activity due to the abnormal level of some immunoglobulins [16]. Osteoarthritis is a degenerative joint disease that affects an animal's quality of life. Osteoarthritis consists of biological and mechanical alterations in articular cartilage synthesis and degradation coupling balance. It is characterized by morphological, biochemical, and molecular changes in articular cartilage chondrocytes, extracellular matrix, and subchondral bone leading to destabilization of normal homeostatic processes. These pathophysiological changes consequently lead to continuous progressive complications including increase bone thickening, deformation of the articular surface, articular edema, and fibrillations [11,12,13]. Musculoskeletal disorders (trauma, arthritis) occur in more than 70% of captive elephants in North America [9]. Non- pharmacological management therapies are not enough to conserve an elephant's bones integrity. According to a survey done by AZA in 2001, regular exercise was found to be beneficial to either reduce the incidence or alleviate
4 osteoarthritic symptoms in captive elephants. However, pharmacological therapy using Non- Steroidal Anti- inflammatory Drugs (NSAIDs) is the cornerstone of treatment of foot trauma and arthritis in captive elephants. NSAIDs are used to treat a variety of conditions in elephants (among 66% of respondent zoos to an international survey of mega- vertebrates kept in zoos) ranging from acute injury pain relief to postsurgical pain management. However, chronic foot problems and arthritis were the most common reason for using NSAIDs in elephants [17,18]. Administration of a safe and effective dose of a drug cannot always be based on interspecies allometric scaling of dosage. The variation in absorption, distribution, metabolism, and elimination among different animals makes the prediction of the doses extremely difficult using extrapolated pharmacokinetic parameters and is often not effective for dosing of different species. Hunter and his colleagues, 2008, used three methods of interspecies scaling including linear extrapolation, metabolic, and allometric scaling. The physiological processes and metabolic rate for mammals can be extrapolated allometrically among species based on the weight of the animal. However, flunixin showed a poor correlation of pharmacokinetic parameters with animal weight when it is administered using allometric scaling method. Another NSAID drug (Phenylbutazone) also was not a good candidate for allometric scaling. Phenylbutazone undergoes extensive metabolism and potentially affected by concurrent food consumption [19-22].
5 Rieviere and his colleagues, 1997, reported that approximately 75% of drugs are not scalable allometrically among species [23]. Even though some pharmacokinetic parameters (clearance, volume of distribution, and half- life) can be extrapolated in large animals, flunixin showed a larger error between predicted and observed values [24]. Drugs that are highly protein bound, extensively metabolized, undergo active transport, and/or are significantly excreted via urine or biliary routes are unlikely to be allometrically scalable among different animal species [19]. African and Asian elephants (Loxodonta africana, Elephas maximus) are entirely different species. They behave differently regarding drug metabolism and clearance. Ibuprofen and phenylbutazone (NSAIDs) showed significantly different pharmacokinetic parameters between the two elephant species [22,25]. Flunixin is highly protein bound drug. Protein binding of flunixin ranged from 87% in goats to more than 99% in cattle [26,27]. In addition, it is actively transported and excreted extensively through the biliary route [28]. Allometric scaling for flunixin meglumine dosing based on the limited data collected from these comparative studies is inadvisable.
6 2- Flunixin meglumine: Flunixin meglumine (Banamine ) is a nicotinic acid derivative (2- methyl- 3- trifluoromethyl- phenyl- amino- nicotinic acid) in the form of n- methylglucamine salt with a pka= 5.82 (weak acid) [29]. It is a non- steroidal anti- inflammatory agent that inhibits cyclooxygenase enzymes (COX) and therefore, inhibits formation of the inflammatory mediators in the arachidonic acid cascade (prostaglandins, prostacyclin, and thromboxane A2). Flunixin meglumine is approved as an antipyretic and analgesic agent for horses [30]. It may also reduce blood dynamic effects caused by E. coli in horses [31]. Flunixin meglumine is available as parenteral (IV, IM) and oral paste dosage forms. It is administered to horses at 1.1 mg/kg of body weight every 12 hours or 2.2 mg/kg of body weight every 24 hours. Flunixin plasma concentration range between 0.2-0.9 μg/ml was determined to provide at least 50% therapeutic efficacy in arthritis treatment [32,33]. Flunixin meglumine may cause untoward adverse events in animals including hematuria in cattle, gastric ulceration in dogs and horses [34,35] especially when it is administered concurrently with phenylbutazone (another non- steroidal anti- inflammatory drug) [36]. Flunixin is metabolized by the liver via hydroxylation enzymes to yield 5- hydroxy- flunixin, which then undergoes glucuronide conjugation to be excreted by kidney (figure 1) [37,38].
7 O OH H N F F F O OH H N F F F N Flunixin HO N 5 -Hydroxyflunixin Conjugate(s) Conjugate(s) Figure 1: Flunixin metabolism in the liver.
8 Flunixin meglumine showed variable pharmacokinetics among different animal models. The pharmacokinetics of flunixin has been extensively studied from the 1980's. The pharmacokinetics of flunixin in cattle was similar to what was found in dogs and horses with two exceptions, half- life was significantly longer, and volume of distribution was greater in cattle [39,40]. The half- life of flunixin in sheep (3.43-3.8 hrs) was consistent with dogs, horses and camels but significantly shorter than cattle. The intramuscular bioavailability was similar to oral bioavailability in cattle and horses [45,46]. Flunixin showed a significantly shorter half- life in cattle in another study, 4 hrs compared to 8.1hrs. A second peak drug concentration was detected 3 hrs after IV administration [41]. Oral bioavailability of flunixin was consistent in cattle from two studies at 53 and 60%. Subcutaneous and Intramuscular administrations showed longer half- lives than intravenous route [43]. Flunixin plasma protein binding was > 98% in cattle [26]. The half- life was shorter in llama (1.47hrs) compared to cattle. The volume of distribution at steady- state (Vdss) was also much smaller in llamas (30ml/kg) compared to cattle (419ml/kg) and camels (320-350ml/kg) while the half- life in camels was consistent with dogs and horses [43,44].
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14 31- Hardie EM, Rawlings CA, Shotts EB, et al. 1987. Escherichia coliinduced lung and liver dysfunction in dogs: effects of flunixin meglumine treatment. Am J Vet Res Jan; 48(1): 56-62. 32- Banamine injectable solution product information (ScheringPlough US), Rev 3/03.Available at www.spah.com. Accessed on December 27, 2006. 33- Lees P, Giraudel J, Landoni MF, Toutain PL. 2004. PK-PD integration and PK-PD modelling of nonsteroidal anti- inflammatory drugs: principles and applications in veterinary pharmacology. Journal of Veterinary Pharmacoloy and Therapeutics.. 27(6):491-502 34- Dow SW, Rosychuk RA, McChesney AE, et al. 1990. Effects of flunixin and flunixin plus prednisone on the gastrointestinal tract of dogs. Am J Vet Res; 51(7): 1131-8. 35- Odensvik K. 1995. Pharmacokinetics of flunixin and its effect on prostaglandin F2 alpha metabolite concentrations after oral and intravenous administration in heifers. Journal of Vet Pharmacol Ther 18: 254-9. 36- Reed SK, NT Messer, RK Tessman and KG Keegan. 2006. Effects of phenylbutazone alone or in combination with flunixin meglumine on blood protein concentrations in horses. American Journal of Veterinary Research 67(3):398-402
15 37- Jaussaud, Ph, Courtot, D. & Guyot, J.J. 1987. Identification of a flunixin metabolite in the horse by gas chromatography±mass spectrometry. Journal of Chromatograhy B, 423: 123-130. 38- Johansson, M. & Anler, E.L.1988. Gas chromatographic analysis of flunixin in equine urine after extractive methylation. Journal of Chromatography, 427: 55-66. 39- Hardee, G.E., Smith, J.A. & Harris, S.J.1985. Pharmacokinetics of flunixin meglumine in the cow. Research in Veterinary Science, 39: 110-112. 40- Hardie, E.M., Hardee, G.E. & Rawlings, C.A. 1985. Pharmacokinetics of flunixin meglumine in dogs. American Journal of Veterinary Research, 46: 235-237. 41- Jedziniak P et al., 2007. Determination of FLUNIXIN and 5-HYDROXYFLUNIXIN in Bovine Plasma with HPLC-UV-Method Development, Validation and Verification. Bull Vet Inst Pulawy 51:261-266 42- Anderson, K. L., C. A. Neff-Davis, and V. D. Bass. 1990. Pharmacokinetics of flunixin meglumine in lactating cattle after single and multiple intramuscular and intravenous administrations. Am. J. Vet. Res. 51:1464 1467.
16 43- Wasfi et al,. 1998. Pharmacokinetics, metabolism and urinary detection time of flunixin after intravenous administration in camels. Journal of Veterinary Pharmacology and Therapeutics. 21, 203-208. 44- Navarre, C. B., W. R. Ravis, R. Nagilla, A. Simpkins, S. H. Duran, and D. G. Pugh. 2001. Pharmacokinetics of phenylbutazone in llamas following single intravenous and oral doses. J. Vet. Pharmacol. Therap 24:227 231. 45- Lacroix, M. Z., V. Gayrard, N. Picard-Hagen, and P. L. Toutain. 2011. Comparative bioavailability between two routes of administration of florfenicol and flunixin in cattle. Rev. Med. Vet. 162:321 324. 46- Welsh EM, McKellar QA, Nolan AM: 1993.The pharmacokinetics of flunixin meglumine in the sheep. J Vet Pharmacol Ther. 16(2):181 188.
17 Chapter 2: High Performance Liquid Chromatography Method, Development, and Validation for the Determination of Flunixin in Asian Elephants plasma SULTAN M. ALSHAHRANI 1,2, URSULA BECHERT 3, JM CHRISTENSEN 1 1- Pharmaceutical Science Department, College of Pharmacy, Oregon State University, Corvallis, OR 2- Clinical Pharmacy Department, College of Pharmacy, King Khalid University, Abha, Saudi Arabia 3- College of Liberal and Professional Studies, University of Pennsylvania, Philadelphia, PA Abstract A simple and efficient high performance liquid chromatography method was developed and validated to determine flunixin concentrations in Asian elephant's (Elephas maximus) plasma. Flunixin was administered orally to an Asian elephant at a dose of 0.8 mg/kg, and blood samples were collected. Flunixin extraction was performed by adding an equal amount of acetonitrile to plasma and centrifuging at 4500 rpm for 25 minutes. The supernatant was removed, and flunixin was analyzed using HPLC- UV detection. Two methods were developed and tested using two mobile phases either with or without adding methanol (ACN: H2O vs. ACN: H2O: MeOH). Both methods showed excellent linearity and reproducibility. The limit of detection was 0.05 μg/ml and limit of quantification was 0.1 μg/ml. Flunixin recovery efficiency was maximized by the addition of methanol to mobile phase (ACN: H2O: MeOH as 50:30:20) at 95% in comparison to 23% without methanol.
18 Introduction Flunixin meglumine (Banamine ) is a nicotinic acid derivative (2 - methyl 3 trifluoromethyl phenyl - amino nicotinic acid) (Figure.1) in the form of meglumine (n- methyl glucamine) salt with a pka = 5.82 (weak acid) [1]. Flunixin is a potent non- steroidal anti- inflammatory agent (NSAID) that inhibits cyclooxygenase enzymes (COX). Flunixin is an approved as antipyretic and analgesic agent for horses. It also may reduce blood dynamic effects caused by E. coli in horses [2-5]. O OH H N F F F N Figure 1: Chemical structure of Flunixin
19 Foot pathology is a real problem in captive elephants. Osteoarthritis is the major concern among musculoskeletal disorders and foot health problems for elephants in North America zoos with one- half of Elephants in captivity showing foot health care problems in zoos. A higher ratio of captive Asian elephants (Elephas maximus) is affected by osteoarthritis than African Elephants according to Association of Zoos and Aquarium (AZA) [5,6,7]. The incidence of arthritis increases as the age of the elephant s increases that often requires therapeutic drug monitoring. Flunixin is the one of most commonly prescribed NSAID agent in elephants [8,9,10]. The available published pharmacokinetic studies (trimithoprime- sulfamethoxazole, metronidazole, succinylcholine, and ampicillin) involve small numbers of elephants due to geographical dispersion of captive elephants populations in North America [11-15]. Flunixin analytical assays in various veterinary animals like cows, horses, rabbits, camels, and llamas have been performed [16-22]. The analytical techniques used to determine flunixin drug concentrations in these animals included techniques such as High Performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC), and Gas Chromatography- Mass Spectroscopy (GC- MS) [16,18,20,23]. An HPLC technique was previously used to detect flunixin in cows and horses since the 80s until to- date [18-19]. GC- MS, on the other hand, was used to evaluate flunixin in horses and camels plasma [17-21].
20 However, HPLC is the most common and convenient analytical technique that determines flunixin in animal's plasma. Reliable flunixin plasma concentrations are achieved by developing an optimized HPLC assay. For example, flunixin is a weakly acidic drug and favorably analyzed using acidic ph (3.1-3.5) of the mobile phase to attain the best chromatographic resolution [16]. Although extraction by evaporation was the most commonly used technique to extract flunixin, it provided moderate flunixin recovery from an animal's plasma [16,18,20,23]. Flunixin Ultra- Violet (UV) absorbance was found to be the highest between 274-290 nm wavelengths [24]. Elephant's plasma provides problems that other animals' plasma do not. In previous studies in our laboratory with Elephant plasma, it was often observed to congeal at room or refrigerated temperature making the analysis of the drug extremely difficult to perform by chromatography [25-26]. Extraction of the drug, (solid or liquid) from the congealed plasma was also difficult as not all substances can be fully removed. Analysts with the latest chromatographic equipment are reluctant to measure drugs from Elephant plasma due to this problem. The main aim of this study is to develop and validate a rapid and efficient bioanalytical assay using high performance liquid chromatography (HPLC) in order to detect and determine flunixin concentrations in Asian elephant plasma according to U.S. Food and Drug Administration (FDA) recommendations [27].
21 Materials and Methods Reagents and Chemicals All reagents and solvents were HPLC grade. Pure Flunixin meglumine powder was purchased from Spectrum (CA, USA) and Diclofenac sodium salt (internal standard) was obtained from Sigma- Aldrich (Germany). Methanol and acetonitrile were purchased from Fisher Scientific (NJ, USA). Acetic acid, which was used to adjust the ph of the mobile phase, was obtained from VWR international (PA, USA). Instrumentation Chromatographic analysis was conducted using a Shimadzu Prominence High Performance Liquid Chromatographic system (Kyoto, Japan model LC- 2010A HT) equipped with UV Detector. Sample centrifugation was carried out using Eppendorf centrifuge 5415C (Brinkmann Instruments, NY, USA). Serum samples were filtered via 0.22 μm nylon filter (VWR International, USA) before injection into HPLC. Method development and designs Two chromatographic methods were evaluated. One was with and the other without the addition of methanol to the mobile phase. The first method consisted of acetonitrile and water at a ratio of 50:50. The second method was with a mobile phase consisting of acetonitrile, water, and methanol in a ratio of 50:30:20. The ph of both studied mobile phases was adjusted to 3.1, and the UV absorbance of flunixin was measured at 278 and 284 nm.
22 Chromatographic conditions HPLC analysis of flunixin was performed with a 4.6 150 mm column (Kinetex, Phenomenex, CA, USA) packed with 5- um C- 18 chromatographic media. Also, it is connected to a pre- column (Security guard 2.1 4.6 mm, Kinetex, Phenomenex, CA, USA) and samples flow through an in- line filter (KrudKatcher ULTRA HPLC, Kinetex, Phenomenex, CA, USA) to further insure removal of impurities. Two mobile phases were studied. The mobile phase of the first method consisted of acetonitrile, water (ph 3.1, pre- adjusted with acetic acid) in the ratio of 50:50 (method I). Methanol was added to the mobile phase of the second method, which consisted of acetonitrile, water (ph 3.1 pre- adjusted with acetic acid), and methanol in the ratio of 50:30:20 (method II). The mobile phases were sonicated for 60 min before use. The flow rate was 0.8 ml/min for the method I, and 0.6 ml/min for method II. The UV- Detector wavelength was set at 278, and 284 nm and the column temperature was set at 25 C. The injection volume was 20 μlfor the method I, and 15 μl for method II respectively. The run time was 12 minutes for the method I, and 8 minutes for method II.
23 Standard solutions and calibration curves preparation A stock solution of Flunixin (1 mg/ml) was prepared in methanol. Standard solutions of flunixin in elephant serum were prepared by adding appropriate volumes of the flunixin (1 mg/ml) stock solution to drug- free elephant serum to produce concentrations of 0.1, 0.5, 1, 5, 10, 20, 40 μg/ml. Each concentration was injected into the HPLC in triplicate utilizing both methods' conditions for flunixin analysis. Three quality control levels were also included at low, middle and high concentrations (1, 5, and 20 μg/ml) and three replicates for each level were also assayed. For calibration curve and quality control analyses, an appropriate amount of elephant's plasma was spiked with the appropriate flunixin stock solution volume in order to achieve the calibration curve range (0.1-40 μg/ml). The average ratios of peak area under the absorbance curve (AUC) of flunixin and diclofenac Na + (Internal standard) from assayed elephant serum were measured and plotted against the corresponding concentration of flunixin in μg/ml to prepare calibration curves for flunixin plasma concentration determination. The slope, intercept, and correlation coefficient (R 2 ) were determined by linear regression analysis for each method. Linearity was checked with F- test lack- of- fit analysis.
24 Sample preparation Blood samples were collected from an Asian Elephant via an ear vein. For the HPLC analysis of flunixin concentrations in the collected elephant plasma samples, 0.5 ml of elephant plasma was pipetted into 2 ml Eppendorf centrifuge tube. For all series of samples (calibrators, quality control levels, and test samples), an equal amount of acetonitrile, and 200μL of internal standard solution (100μg/ml Diclofenac sodium) were added. Then, the contents of the tube were mixed for 1 min on a vortex mixer and then allowed to sit for 15 min to denature. After that, the mixture was centrifuged at 4500 rpm 25 min. The supernatant was filtered and transferred to a new HPLC glass tube. The previously determined volume for each method was injected into the HPLC.
25 Results Appropriateness of chromatographic conditions The addition of methanol to the method II (ACN: H2O: MeOH) gave better separation between flunixin and internal standard than the method I (ACN: H2O). Retention times of flunixin and diclofenac were about 6.2 and 9 minutes with mobile phase in the method I, and 6.5 and 7.5 minutes with mobile phase in method II (Figure. 2,3). Flunixin optimal UV absorbance was at 278 nm. Figures 4 and 5 report the chromatogram of elephant serum without flunixin but with diclofenac. Complete separation was observed between plasma components, flunixin, and diclofenac for method II. A small shoulder, apparently from a substance in plasma, overlapped with flunixin in method I (at 5.8 min). Method validation The validity of the HPLC assay method was performed according to FDA recommendations for assessment. The validity of proposed HPLC assay methods included the assay's linearity, LOD, LOQ, accuracy, precision, and selectivity.
Figure 2: Method II (50:20:30) elephant plasma spiked with 10 µg/ml Flunixin and internal standard (Diclofenac Na + ) 15μL injection volume. 26
Figure 3: Method I (50:50) elephant plasma spiked with 10 µg/ml flunixin and internal standard (Diclofenac Na + ) 15μL injection volume 27
Figure 4: Method I (50:50) Blank elephant plasma with internal standard (Diclofenac Na + ) 15μL injection volume. 28
Figure 5: Method II (20:30:50) Blank elephant plasma with internal standard (Diclofenac Na + ) 15μL injection volume. 29
30 Linearity For the previous two described HPLC methods, an excellent linear relationship was found. Plotting the average chromatograms ratio of peak areas (AUC) of flunixin to the internal standard's AUC versus concentration range (0.1 40 μg/ml) a straight line was observed (Figure 6,7). The concentration range contains 7 points for both methods. Linear regression on the calibration curves were performed, and one- way ANOVA test was used to calculate the slope and intercept for method I (Y= 0.017 X + 0.0007, and R 2 = 0.9996), and for method II (Y= 0.0186 X + 0.0128, and R 2 = 0.997); Where Y is absorption peak area ratio (flunixin AUC: Internal standard AUC), C is the corresponding concentration (μg/ml), and R 2 is correlation coefficient (Figure. 7). The high R- square value (R 2 ) indicates the goodness- of- fit of the calibration curves' linearity for both methods.
31 ACN:Water 50:50 0.8 0.7 y = 0.017x + 0.0007 R² = 0.99966 0.6 AUC Ratio (FXN:IS) 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 45 Concentration (ug/ml) Figure 6: Calibration curve of Method I ACN:Water at ratio of 50:50
32 ACN:MeOH:Water 50:20:30 0.9 0.8 y = 0.0186x + 0.0128 R² = 0.99794 0.7 AUC Ratio (FXN:IS) 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 45 Concentration (ug/ml) Figure 7: Calibration curve of Method II ACN:Methanol:Water at ratio of 50:20:30
33 Limit of quantification (LOQ) & Limit of detection (LOD) The lower limit that can be reliably detected by both HPLC methods' conditions was 0.05 μg/ml (LOD). The limit of quantification below that the flunixin concentration cannot be measured is 0.1 μg/ml. The calibration curve shows a non- linear pattern below 0.1 μg/ml (LOQ). Accuracy Performing three replicate analyses at three concentration levels (quality controls) 1, 5, 20 μg/ml on three consecutive days were done to assess intra- day and inter- day recovery of flunixin from the proposed two methods. Method I, showed very low recovery rate (inter- day, and intra- day) of flunixin from elephant's plasma compared to method II. The flunixin inter- day recovery percentage with the addition of methanol (method II) was between 94.5 96.4% in comparison to 22.8 26.94% from method I (without methanol). The intra- day recovery rate of flunixin from the method I (without methanol) was between 23.2 31.4 % in comparison to 93.4 94.7 % with method II (with an addition of 20% methanol) Tables.1, 2. Precision The coefficient of variation (%CV) was calculated for the averages of peak areas ratio (FXN: IS) for three flunixin elephant plasma concentrations 1, 5, 20 μg/ml. The intra- day %CV was between 0.7 1.3 for both methods, and the inter- day %CV was between 0.16 3.7 for both methods (Table. 1, 2).
34 Method I: ACN:Water (50:50) Concentration (µg/ml ) Interday Accuracy (% ±SD) Intraday Accuracy (% ± SD) Low 1 22.8 ± 1.9 23.23 ± 1.0 Intermediate 5 28.95 ± 0.01 31.4 ± 3.12 High 20 26.94 ± 0.0003 27.1 ± 98 Concentration (µg/ml ) Interday Precision (% CV) Intraday Precision (% CV) Low 1 0.16 0.81 Intermediate 5 0.19 0.9 High 20 0.45 0.714 Table 1: Method I Accuracy and Precision
35 Method II: ACN:MeOH:Water (50:20:30) Concentration (µg/ml) Interday Accuracy (% ±SD) Intraday Accuracy (% ± SD) Low 1 94.59 ± 0.0007 94.5 ± 0.0001 Intermediate 5 96 ± 0.004 94.7 ± 0.0016 High 20 94.5 ± 0.098 93.4 ± 0.014 Concentration (µg/ml ) Interday Precision (% CV) Intraday Precision (% CV) Low 1 3.7 1.33 Intermediate 5 1.63 1.04 High 20 0.5 0.71 Table 2: Method II Accuracy and Precision