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The disposition of fluoxetine in newborn lambs up to one year of age Chow, Timothy W. Y. 2013

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The Disposition of Fluoxetine in Newborn Lambs Up to One Year of Age by Timothy W.Y. Chow B.Sc., University of Victoria, 2006 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) March 2013  © Timothy W.Y. Chow 2013  Abstract	
   While drugs are commonly used in the pediatric population, their disposition is not well studied. The passive transfer of drugs administered to pregnant women to the fetus constitutes a major route of drug exposure in newborns. The fetus has maternal routes for drug disposition; however, newborns must rely on their own immature mechanisms. Therefore, a thorough understanding of the ability of newborns to absorb, distribute, metabolize. and excrete drugs, and the ontogeny of drug disposition is essential to determine the development of these mechanisms from birth to adult life with the goal of providing clinicians with guidelines for optimal drug dosing.  The lifetime incidence of depression in women is approximately 10-25% with a greater vulnerability for the onset or recurrence of depression during childbearing years. Approximately 30-35% of pregnant women diagnosed with depression are on an antidepressant, such as the selective serotonin reuptake inhibitors (SSRI). One of the most prescribed SSRIs that is also used during pregnancy is fluoxetine. The prevalence of SSRI use during pregnancy results in 5% of all newborns in our population with antenatal SSRI exposure. Stereoselective disposition of fluoxetine has been demonstrated in sheep and humans.  This thesis utilized the chronically catheterized lamb animal model to study fluoxetine disposition ability from newborn to one year of life. This model builds upon previous research in our group that used pregnant, non-pregnant, and newborn sheep to  ii  characterize the maternal, fetal, and neonatal disposition of clinically relevant compounds. The studies in this thesis on postnatal fluoxetine disposition build upon previous research on maternal-fetal fluoxetine stereoselective disposition in our group. These studies reported no metabolism of fluoxetine in the fetus, and their disposition depended on maternal routes.  The disposition of fluoxetine in newborn lambs was limited compared to adults but was developed compared to the fetus. A gender difference was observed in the newborn group where females had a greater metabolic capacity for fluoxetine. A significant maturation in drug disposition was observed at approximately 3 months, which coincided with the time of weaning. Fluoxetine disposition was stereoselective from newborn to one year of age.  iii  Preface	
   All use of animals in this study conformed to the guidelines of the Canadian Council on Animal Care and were approved in advance by the University of British Columbia Committee on Animal Care (A07-0302). The enantioselective LC/MS/MS assay described in section 2.3.1 that was used to quantify fluoxetine and norfluoxetine levels in plasma samples was published in the Journal of Chromatography. Chow, TW et. al. 2010. “A Validated Enantioselective Assay for the Simultaneous Quantitation of (R)-,(S)-Fluoxetine and (R)-,(S)-Norfluoxetine in Ovine Plasma Using Liquid Chromatography with tandem mass spectrometry” Journal of Chromatography B.879:349-359. At the time of writing this thesis, the outcomes of these studies have not been published. A manuscript on the development of fluoxetine disposition in postnatal lambs up to 1 year of age, and another on sex differences in fluoxetine disposition in postnatal lambs up to 1 year of age are planned and will be submitted for publication.  iv  Table	
  of	
  contents	
  	
   Abstract	
  .......................................................................................................................................	
  ii	
   Preface	
  ........................................................................................................................................	
  iv	
   Table	
  of	
  contents	
  .....................................................................................................................	
  v	
   List	
  of	
  tables	
  ............................................................................................................................	
  vii	
   List	
  of	
  figures	
  ............................................................................................................................	
  ix	
   List	
  of	
  abbreviations	
  ...........................................................................................................	
  xiii	
   Acknowledgements	
  ............................................................................................................	
  xvii	
   1.	
   Introduction	
  ......................................................................................................................	
  1	
   1.1.1	
   Physicochemical	
  properties	
  ........................................................................................................	
  3	
   1.1.2	
   Pharmacology	
  and	
  therapeutic	
  uses	
  ........................................................................................	
  3	
   1.1.3	
   Pharmacokinetics	
  ADME	
  ..............................................................................................................	
  5	
   1.1.4	
   Stereoselective	
  properties	
  of	
  fluoxetine	
  and	
  norfluoxetine	
  .......................................	
  12	
   1.2	
   Depression	
  and	
  pregnancy	
  ...............................................................................................	
  13	
   1.2.1	
   Incidence	
  of	
  depression	
  during	
  pregnancy	
  .......................................................................	
  13	
   1.2.2	
   Antidepressant	
  use	
  during	
  pregnancy	
  .................................................................................	
  14	
   1.2.3	
   SSRI	
  use	
  during	
  pregnancy	
  and	
  birth	
  outcomes	
  ..............................................................	
  16	
   1.3	
   Pediatric	
  drug	
  dosing	
  and	
  disposition	
  .........................................................................	
  20	
   1.3.1	
   Lack	
  of	
  knowledge	
  ........................................................................................................................	
  20	
   1.3.2	
   Current	
  research	
  ...........................................................................................................................	
  22	
   1.3.3	
   Ontogeny	
  of	
  drug	
  disposition	
  ..................................................................................................	
  23	
   1.4	
   Study	
  rationale	
  and	
  experimental	
  model	
  ....................................................................	
  24	
   1.5	
   Research	
  objectives	
  and	
  hypothesis	
  .............................................................................	
  26	
   2	
   Methods	
  .............................................................................................................................	
  27	
   2.1	
   Experimental	
  model	
  ...........................................................................................................	
  27	
   2.1.1	
   Breeding	
  ............................................................................................................................................	
  28	
   2.1.2	
   Surgical	
  preparation	
  ....................................................................................................................	
  29	
   2.1.3	
   Routine	
  maintenance	
  ..................................................................................................................	
  30	
   2.2	
   Experimental	
  protocols	
  .....................................................................................................	
  30	
   2.2.1	
   Fluoxetine	
  preparation	
  ...............................................................................................................	
  30	
   2.2.2	
   Urine	
  collection	
  ..............................................................................................................................	
  31	
   2.2.3	
   Pharmacokinetic	
  experiments	
  ................................................................................................	
  33	
   2.3	
   Analytical	
  methods	
  .............................................................................................................	
  34	
   2.3.1	
   LC/MS/MS	
  assay	
  for	
  plasma	
  samples	
  ..................................................................................	
  34	
   2.3.2	
   LC/MS/MS	
  assay	
  for	
  urine	
  samples	
  ......................................................................................	
  46	
   2.3.3	
   Plasma	
  protein	
  binding	
  ..............................................................................................................	
  55	
   2.3.4	
   Alpha-­‐1	
  Acid	
  Glycoprotein	
  immunoassay	
  ..........................................................................	
  58	
   2.3.5	
   Bradford	
  immunoassay	
  ..............................................................................................................	
  58	
   2.3.6	
   Blood	
  pH	
  and	
  temperature	
  .......................................................................................................	
  59	
   2.4	
   Pharmacokinetic	
  analyses	
  ...............................................................................................	
  59	
   2.5	
   Statistical	
  analysis	
  ...............................................................................................................	
  61	
   3.	
   Results	
  ..............................................................................................................................	
  63	
   3.1	
   Stereoselective	
  fluoxetine	
  disposition	
  in	
  newborns	
  to	
  1	
  year	
  of	
  age.	
  ...............	
  63	
   3.1.1	
   Developmental	
  plasma	
  fluoxetine	
  pharmacokinetics	
  ...................................................	
  63	
   3.1.2	
   Stereoselective	
  plasma	
  pharmacokinetics	
  .........................................................................	
  76	
   3.1.3	
   Developmental	
  urinary	
  pharmacokinetics	
  ........................................................................	
  82	
   v  3.1.4	
   Stereoselective	
  urinary	
  pharmacokinetics	
  ........................................................................	
  88	
   3.1.5	
   Developmental	
  and	
  stereoselective	
  fluoxetine	
  and	
  norfluoxetine	
  plasma	
   protein	
  binding	
  ..............................................................................................................................................	
  92	
   3.1.6	
   Developmental	
  plasma	
  protein	
  levels	
  ..................................................................................	
  96	
   3.1.7	
   Sex	
  differences	
  in	
  fluoxetine	
  disposition	
  from	
  newborn	
  to	
  1	
  year	
  of	
  age.	
  ...........	
  99	
   3.1.8	
   Stereoselective	
  urinary	
  data	
  compared	
  between	
  sex	
  ................................................	
  105	
   3.1.9	
   Fluoxetine	
  and	
  norfluoxetine	
  plasma	
  protein	
  binding,	
  blood	
  pH	
  and	
   temperature	
  compared	
  between	
  sex	
  ................................................................................................	
  114	
   3.1.10	
   Plasma	
  protein	
  levels,	
  temperature	
  and	
  pH	
  compared	
  between	
  sex	
  ..................	
  116	
    4.	
   Discussion	
  .....................................................................................................................	
  120	
   4.1	
   Ontogeny	
  of	
  fluoxetine	
  disposition	
  .............................................................................	
  120	
   4.2	
   Stereoselective	
  fluoxetine	
  and	
  metabolite	
  disposition	
  ........................................	
  133	
   4.3	
   Fluoxetine	
  and	
  norfluoxetine	
  plasma	
  protein	
  binding	
  and	
  developmental	
   plasma	
  protein	
  levels.	
  .................................................................................................................	
  139	
   4.4	
   Sex	
  differences	
  in	
  fluoxetine	
  disposition	
  ..................................................................	
  142	
   4.5	
   Conclusions	
  ..........................................................................................................................	
  149	
   Bibliography	
  .........................................................................................................................	
  151	
   Appendices	
  ...........................................................................................................................	
  174	
   Appendix	
  1	
  Plasma	
  fluoxetine	
  pharmacokinetic	
  data	
  of	
  individual	
  lambs	
  ..............	
  174	
   Appendix	
  2	
  Urine	
  data	
  for	
  individual	
  lambs	
  .......................................................................	
  188	
   Appendix	
  3:	
  Cohorts	
  1	
  to	
  6	
  blood	
  pH	
  and	
  body	
  temperature	
  data.	
  .............................	
  200	
    	
    vi  List	
  of	
  tables	
   Table 2.1 Nomenclature used to classify lamb age groups from newborn to ~1 year of age. ................................................................................................................... 28	
   Table 2.2 Intra-day and inter-day accuracy and precision results for the validation of fluoxetine enantiomers. .................................................................................... 41	
   Table 2.3 Intra-day and inter-day accuracy and precision results for the validation of norfluoxetine enantiomers. ............................................................................... 42	
   Table 2.4 Intra-day and inter-day accurayc and precision results for the validation of fluoxetine enantiomers ..................................................................................... 51	
   Table 2.5 Intra-day and inter-day accuracy and precision results for the validation of norfluoxetine enantiomers. ............................................................................... 52	
   Table 2.6 Intra-day and inter-day accuracy and precision results for the validation of TFMP................................................................................................................ 52	
   Table 2.7 Urine LC/MS/MS assay matrix effect validation results. ................................. 52	
   Table 2.8 Urine LC/MS/MS assay dilution integrity and %carry-over validation results.53	
   Table 3.1 Average weight (kg) and age (days) of lambs in cohorts 1 to 6 at the start of pharmacokinetic experiments. .......................................................................... 63	
   Table 3.2 Fluoxetine plasma pharmacokinetic data for newborns (Cohorts 1) to ~1 Month of age (Cohort 3) (AVG ± SEM)...................................................................... 70	
   Table 3.3 Fluoxetine plasma pharmacokinetic data compared between ~1 month (Cohort 3) and ~3 months of age (Cohort 4) (AVG ± SEM). ....................................... 71	
   Table 3.4 Fluoxetine and norfluoxetine plasma pharmacokinetic data at ~3 months (Cohort 4) to ~ 12 months of age (Cohort 6) (AVG ± SEM). .......................... 72	
   Table 3.5 Fluoxetine systemic clearance as a function of postnatal age using piecewise regression analysis with 2-elements (AVG ± SEM). ....................................... 73	
   Table 3.6 Piecewise regression analysis with 2-elements of plasma NFX/FX AUC ratio as a function of postnatal age (AVG ± SEM). ................................................. 75	
   Table 3.7 Stereoselective free clearance for cohorts 1 to 6. ............................................. 95	
   Table 3.8 Average weight (kg) and age (days) of lambs in cohorts 1 to 6 at the start of pharmacokinetic experiments (AVG ± SEM). ................................................. 99	
   Table 3.9 Male and female newborn (Cohort 1) fluoxetine plasma pharmacokinetic data (AVG ± SEM). ............................................................................................... 101	
   Table 3.10 Plasma pharmacokinetics data for males and females in cohorts 1 to 6 (AVG ± SEM). ............................................................................................................. 102	
   Table 3.11 Break point, slopes, and intercept values using piecewise 2-element regression analysis of the relationship between fluoxetine systemic clearance and postnatal age for male and female lambs (AVG ± SEM)............................... 104	
   Table 3.12 Fluoxetine free clearance values for males and females in cohorts 1 to 6.... 115	
   Table 4.1 Fluoxetine pharmacokinetic parameters after i.v. bolus administration of racemic fluoxetine in fetal, pregnant, newborn and adult non-pregnant sheep (AVG ± SD). .................................................................................................. 120	
    vii  Table 4.2 Fetal and maternal placental and non-placental clearance values after i.v. bolus administration of racemic fluoxetine in separate studies to the fetus and mother. (Kim 2000) ........................................................................................ 122	
   Table 4.3 Stereoselective formation of norfluoxetine in adult and fetal sheep measured by in vitro microsome studies.(Kim 2000) .......................................................... 135	
   Table 4.4 Stereoselective formation of norfluoxetine using human cDNA-expressed CYP isozyme microsomes.(Kim 2000)................................................................... 136	
    viii  List	
  of	
  figures	
   Figure 1.1 The 3-dimensional chemical structures of fluoxetine enantiomers presented as mirror images of each other................................................................................ 3	
   Figure 1.2 A schematic diagram of fluoxetine’s proposed Phase I and II metabolic pathways. ......................................................................................................... 9	
   Figure 1.3 A proposed kinetic equation describing the mass transfer process of drug to plasma protein binding. ...................................Error! Bookmark not defined.	
   Figure 2.1 Schematic drawing of the male lamb urethra. ................................................. 31	
   Figure 2.2 Urine strap and collection bag arrangement used to collection urine from male lambs. ............................................................................................................... 32	
   Figure 2.3 Proposed daughter fragments used for multiple reaction monitoring analysis of fluoxetine and norfluoxetine. ........................................................................... 36	
   Figure 2.4 Representative product ion mass spectra (ESI+) of (R)-,(S)-fluoxetine (a), and (R)-,(S)-norfluoxetine (b). ................................................................................ 37	
   Figure 2.5 Limit of quantitation and selectivity in ovine plasma of racemic fluoxetine in blank ovine plasma (a), racemic fluoxetine in blank ovine plasma (b), racemic fluoxetine 1 mg/ml standard in ovine plasma (c), and racemic norfluoxetine 1 ng/ml standard in ovine plasma. .................................................................... 44	
   Figure 2.6 Proposed fragmentation ions used for multiple reaction monitoring analysis of TFMP. ............................................................................................................ 48	
   Figure 2.7 Ion mass spectra for the multiple reaction monitoring analysis of TFMP (a), TFMP internal standard (b), fluoxetine enantiomers (c), norfluoxetine enantiomers (d), and d5-fluoxetine enantiomers............................................ 53	
   Figure 2.8 Limit of quantitation and selectivity in ovine urine blank (a), and 1 ng/ml standard in ovine urine (b). ............................................................................ 54	
   Figure 2.9 A schematic equation showing the hydrolysis reaction of β-Glucuronidase .. 54	
   Figure 2.10 Schematic diagrma of the mechanism of equilibrium dialysis. ..................... 57	
   Figure 3.1 Postnatal weight (kg) plotted as a function of age (days) of the lambs from newborn (Cohort 1) to ~1 year of age (Cohort 6). ......................................... 64	
   Figure 3.2 Newborn (Cohort 1) average plasma fluoxetine concentrations on logarithmic scale as a function of time. Log scale was used for fluoxetine concentrations to best show the tri-phasic decline (AVG ± SEM). ....................................... 65	
   Figure 3.3 Cohort 1 average fluoxetine and norfluoxetine plasma concentrations plotted as a function of time (AVG ± SEM). ............................................................. 67	
   Figure 3.4 Cohort 2 average fluoxetine and norfluoxetine concentrations plotted as a function of time (AVG ± SEM). ...................................................................... 67	
   Figure 3.5 Cohort 3 average fluoxetine and norfluoxetine plasma concentrations plotted as a function of time (AVG ± SEM). ............................................................. 68	
   Figure 3.6 Cohort 4 average fluoxetine and norfluoxeitne plasma concentrations plotted as a function of time (AVG ± SEM). ............................................................. 68	
   Figure 3.7 Cohort 5 average fluoxetine and norfluoxetine plasma concentrations plotted as a function of time (AVG ± SEM). ............................................................. 69	
    ix  Figure 3.8 Cohort 6 average fluoxetine and norfluoxetine plasma concentrations plotted as a function of time (AVG ± SEM). ............................................................. 69	
   Figure 3.9 Fluoxetine systemic clearance as a function of post-natal age. The regression line was estimated using piecewise regression with 2-elements.................... 73	
   Figure 3.10 Fluoxetine volume of distribution at steady state as a function of post-natal age. The line was estimated linear regression (r 2= -0.474, p = 0.745).......... 74	
   Figure 3.11 Plasma NFX/FX AUC ratio as a function of postnatal age. The regression line was estimated using piecewise linear regression with 2-elements. ........ 75	
   Figure 3.12 Plasma metabolite to parent compound ratio (NFX/FX) clearance. The line was estimated using linear regression (r 2= 0.786, p < 0.001). ...................... 76	
   Figure 3.13 Newborn (Cohort 1) S/R ratio of plasma pharmacokinetic data (AVG ± SEM); the asterisk (*) denotes significant difference from 1.. ...................... 77	
   Figure 3.14 Average plasma fluoxetine enantiomer concentrations as a function of time in newborns (AVG ± SEM). .............................................................................. 78	
   Figure 3.15 Cohort 2 S/R ratio of plasma pharmacokinetic data (AVG ± SEM); the asterisk (*) denotes significant difference from 1. ......................................... 79	
   Figure 3.16 Cohort 3 S/R ratio of plasma pharmacokinetic data (AVG ± SEM); the asterisk (*) denotes significant difference from 1. ...................................... 80	
   Figure 3.17 Cohort 4 S/R ratio of plasma pharmacokinetic data (AVG ± SEM); the asterisk (*) denotes significant difference from 1 .......................................... 80	
   Figure 3.18 Cohort 5 S/R ratio of plasma pharmacokinetic data (AVG ± SEM), the asterisk (*) denotes significant difference from 1.. ........................................ 81	
   Figure 3.19 Cohort 6 S/R ratio of plasma pharmacokinetic data (AVG ± SEM), the asterisk (*) denotes significant difference from 1. ......................................... 81	
   Figure 3.20 Systemic clearance S/R ratio as a function of postnatal age. The regression line was estimated using linear regression (r = -0.032, p 0.496). .................. 82	
   Figure 3.21 Fluoxetine weight adjusted renal clearance from newborn (Cohort) 1 to ~1 year of age (Cohort 6). Letters a and b denotes significant differences (AVG ± SEM). ....................................................................................................... 83	
   Figure 3.22 Representative urine fluoxetine and TFMP levels as a function of time in newborn lambs (AVG ± SEM). ................................................................... 84	
   Figure 3.23 Fluoxetine and Phase I metabolites norfluoxetine and TFMP concentrations in urine samples as a function of time at ~ 1 month of age (Cohort 3) (AVG ± SEM). ....................................................................................................... 85	
   Figure 3.24 Fluoxetine and norfluoxetine glucuronide concentrations in urine as a function of time at ~1 month of age (Cohort 3) (AVG ± SEM). ................... 86	
   Figure 3.25 Comparison of cummulative urine levels of fluoxetine and its metabolites between ~1 month (Cohort 3) an ~ 3 months (Cohort 4) (AVG ± SEM). The asterisk (*) denotes significant differences .................................................... 87	
   Figure 3.26 Amount recovered in urine for fluoxetine, norfluoxetine, fluoxetineglucuronide, and norfluoxetine-gluruconide compared from ~1 month (Cohort 3) to ~12 months (Cohort 6). The asterisk (*) denotes significant difference (AVG ± SEM). .............................................................................. 88	
   Figure 3.27 Representative urine concentrations of fluoxetine enantiomers as a function of time in newborns (AVG ± SEM). .............................................................. 89	
    x  Figure 3.28 Representative urine fluoxetine and norfluoxetine enantiomers, and TFMP concentrations as a function of time. at ~ 1 month (Cohort 3) (AVG ± SEM). ........................................................................................................... 90	
   Figure 3.29 Representative urine fluoxetine and norfluoxetine enantiomer glucuronide concentrations as a function of time at ~ 1 month (Cohort 3) (AVG ± SEM). ........................................................................................................................ 91	
   Figure 3.30 S/R ratio of urine pharmacokinetic parameters compared between cohorts 1 to 6 (AVG ± SEM) ......................................................................................... 92	
   Figure 3.31 Unbound fraction of fluoxetine in plasma for cohorts 1 to 6; Columns with different letters above them are significantly different (AVG ± SEM). ...... 93	
   Figure 3.32 S/R ratio of unbound fraction of fluoxetine in plasma for cohorts 1 to 6 (AVG ± SEM). The asterisk (*) denotes statistical significance from null hypothesis of 1. ............................................................................................ 94	
   Figure 3.33 Norfluoxetine fraction unbound in plasma for cohorts 1 to 6Columns with different letters above them are significantly different based on two-way ANOVA (age, gender) (AVG ± SEM).. ........................................................ 95	
   Figure 3.34 S/R ratio of norfluoxetine unbound fraction for Cohorts 1 to 6. (AVG ± SEM). The asterisk (*) denotes statistical significance from null hypothesis of 1. Sample size: Cohorts 1 to 2 = 7; Cohort 3 = Pooled; Cohort 4 to 6 = 6). .. 96	
   Figure 3.35 AAG plasma concentrations for cohorts 1 to 6; Columns with different letters above them are significantly different based on two-way ANOVA (age, gender) (AVG ± SEM). .................................................................................. 97	
   Figure 3.36 Total plasma protein concentrations for cohorts 1 to 6; Columns with different letters above them are significantly different based on two-way ANOVA (age, gender) (AVG ± SEM) .......................................................... 98	
   Figure 3.37 Postnatal weight (kg) as a function of age (days) for male and female lambs. ................................................................................................................... 100	
   Figure 3.38 Average plasma fluoxetine concentrations as a function of time for male and female newborn lambs (AVG ± SEM). ..................................................... 101	
   Figure 3.39 Piecewise regression analysis of fluoxetine clearance as a function of postnatal age. ............................................................................................. 104	
   Figure 3.40 Representative fluoxetine concentrations in urine samples of male and female newborn lambs. .......................................................................................... 106	
   Figure 3.41 Renal excretion of FX compared between males and females in cohorts 1 to 6 (AVG ± SEM). The asterisk (*) denotes significant gender differences. .... 106	
   Figure 3.42 Fluoxetine weight-adjusted renal clearance compared between males and females in cohorts 1 to 6 (AVG ± SEM). The asterisk (*) denotes significant gender differences. ....................................................................................... 107	
   Figure 3.43 Fluoxetine renal clearance plotted as a function of systemic clearance for all lambs. ......................................................................................................... 108	
   Figure 3.44 Representative TFMP concentrations in urine samples for male and female newborn lambs. .......................................................................................... 109	
   Figure 3.45 TFMP urinary accumulation compared between males and females in cohorts 1 to 6 (AVG ± SEM). ................................................................................ 109	
   Figure 3.46 NFX urinary accumulation in Cohorts 3 to 6 compared between genders (AVG ± SEM). ............................................................................................. 110	
    xi  Figure 3.47 FX-glucuronide urinary accumulation in Cohorts 3 to 6 compared between genders (AVG ± SEM). ............................................................................. 111	
   Figure 3.48 NFX-glucuronide urinary accumulation in Cohorts 3 to 6 compared between genders. (AVG ± SEM). ............................................................................ 111	
   Figure 3.49 S/R ratio of FX weight-adjusted renal clearance compared between genders for cohorts 1 to 6 (AVG ± SEM) ............................................................... 112	
   Figure 3.50 S/R ratio of the amount of fluoxetine recovered in urine compared between genders for cohorts 1 to 6 (AVG ± SEM). ................................................ 113	
   Figure 3.51 S/R ratio of NFX urinary accumulation in urine for cohorts 1 to 6 (AVG ± SEM). ......................................................................................................... 113	
   Figure 3.52 Unbound fraction of fluoxetine in plasma for male and female lambs from cohort 1 to 6 (AVG ± SEM)......................................................................... 114	
   Figure 3.53 Unbound fraction of norfluoxetine compared between age and gender (AVG ± SEM). ..................................................................................................... 116	
   Figure 3.54 Plasma AAG concentration compared by age and gender (AVG ± SEM). 117	
   Figure 3.55 Blood pH of newborn male and female lambs (AVG ± SEM). .................. 119	
   Figure 4.1 Comparison of fluoxetine plasma pharmacokinetics between fetal, newborn, maternal, and adult sheep after i.v. bolus administration of racemic fluoxetine for clearance (a), elimination half-life (b), and volume of distribution at steady state (c). (Kim 2000) ......................................................................... 121	
   Figure 4.2 Plasma fluoxetine concentration in postnatal lambs as a function of the time since the last maternal FX dose after in utero exposure to chronic maternal fluoxetine dosing during pregnancy............................................................. 124	
   Figure 4.3 Relative hepatic mRNA levels of CYP2C19, CYP2D6, and HNF4a in fetuses treated with saline or cortisol as well as in newborns, and adult sheep.(Pretheeban 2012) .............................................................................. 126	
   Figure 4.4 Relative hepatic mRNA levels of UGT1A6(a), UGT1A9 (b), UGT 2B7 (c), and HNF4α. Measured values from each experimental model is presented as solid circles, mean value +/- SEM given by open diamonds, different letters indicate significant differences. (FS, fetuses treated with saline; FC fetuses treated with cortisol; NB, untreated newborn lambs; AD, untreated adult sheep)(Pretheeban 2011) .............................................................................. 127	
   Figure 4.5 AAG levels from study by Wood et. al. 1986 where increasing levels of alpha1-acid glycoprotein (AAG) in plasma were observed as a function of age.(Wood 1981).......................................................................................... 140	
    xii  List	
  of	
  abbreviations	
   µ  Micron  n  Nano  °C  Degree Celsius  µg  Microgram  µm  Micrometer  µM  Micromolar  ng  Nanogram  nl  Nanoliter  nm  Nanometer  nM  Nanomolar  ~  Approximately  AAG  α1-acid glycoprotein  ADME  Absorption, distribution, metabolism, elimination  AIC  Akaike’s Information Criterion  ANOVA  Analysis of variance  AUC  Area under the concentration vs. time curve  β  Terminal elimination rate constant  CLH  Hepatic clearance  CLf  Fetal total clearance  CLfn  Fetal non-placental clearance  CLfp  Fetal placental clearance  xiii  CLfm  Placental clearance from the fetus to the mother  Clint  Intrinsic clearance  CLm  Maternal total clearance  CLmn  Maternal non-placental clearance  CLmp  Maternal placental clearance  CLrenal  Renal clearance of the total drug  CYP  Cytochrome P-450 enzyme  Da  Dalton  EM  Extensive metabolizer  FAUC  Fetal drug AUC  F/M  Fetal-to-maternal  FX  Fluoxetine  g  Gram  GC  Gas chromatography  GC-MS  Gas Chromatography Mass Spectrometry  HNF4α  Hepatocyte nuclear factor 4 alpha  hr  Hour  HPLC  High performance liquid chromatography  i.e.  id est; that is  i.v.  Intravenous  kg  Kilogram  l  Liter  LOQ  Limit of quantitation  xiv  M  Molar (moles/litre)  MAUC  Maternal drug AUC  mg  Milligram  min  Minute  ml  Milliliter  mm  Millimeter  mM  Millimolar  MRM  Multiple Reaction Monitoring  MRT  Mean residence time  MS  Mass spectrometry  MW  Molecular weight  n  Sample size  NFX  Norfluoxetine  ng  Nanogram  nmol  Nanomole  PBS  Phosphate-buffered saline  pH  Negative logarithm of hydrogen ion concentration  PK  Pharmacokinetic  pKa  Negative logarithm of acid association constant  PM  Poor metabolizer  pmol  picomole  QC  Quality control  QH  Hepatic blood flow  xv  r2  Coefficient of determination  RFX  R enantiomer of fluoxetine  RNFX  R enantiomer of norfluoxetine  RSD  Relative standard deviation  SC  Schwarz Criteron  S/R  S to R ratio  SD  Standard Deviation  SFX  S enantiomer of fluoxetine  SNFX  S enantiomer of norfluoxetine  SSRI  Selective serotonin reuptake inhibitor  t1/2 β  Apparent terminal elimination half-life  TBEP  Tris-bitoxy ethyl phosphate  TFMP  Trifluoromethylphenol  TFMP-IS  2-Chloro-3-(trifluoromethyl)phenol;internal standard used to quantitate Trifluoromethylphenol  tmax  Time of maximum concentration  UGT  UDP-glycuronosyltransferase  Vd  Volume of distribution  VDss  Apparent steady-state volume of distribution  Xu  Cumulative amount of drug or metabolite collected in urine  xvi  Acknowledgements	
   I would like to acknowledge Drs. Dan Rurak and Wayne Riggs for the opportunity and support to be a part of their research. Their passion and sagacity for research is admirable and an inspiration in my growth as a scientist. My sincere gratitude to my graduate research committee members Drs. Mary Ensom, Stelvio Bandiera, Tim Oberlander, and David Grierson, and external examiners Drs. Emma Guns, Thomas Chang, and Pollen Yeung for their valuable time and critical insights in guiding my training.  Thank you to Dr. John Kim, Dr. Caly Chien, Manoja Pretheeban MSc and TuanAhn Nguyen PhD candidate whose dissertation works enriched my studies. Special thanks to the exceptional CFRI staffs Claire Harrison, Edward Zhu, Leika LibeVidanelage, and German Hernandez for their camaraderie and help over the years. Thank you to Dr. Wasan and his group for the use of their equipment, and Dr. Kathy Saatchi for her help in synthetic chemistry. Lastly, thank you to Rachel Wu, Dr. Barb Conway, Wes Wong, and Jonathan Van Drunen for their assistance over the years.  My achievements would not be possible without the unwavering support and encouragement from my family and friends to whom I dedicate this thesis. They give me strength for my endeavors and never let me forget that no matter where I am their support is never far.  xvii  1.  Introduction	
   The disposition of many drugs in infant life is not well studied.(De Cock et al. 2011)  Drug therapy is commonly used in the pediatric population, but passive transfer of drugs taken by the mother to the fetus in utero constitutes a major route of drug exposure for newborns.(Moore et al. 2002) In utero, the fetus has maternal routes for drug disposition; however, after birth newborns must rely on their own immature mechanisms. The disposition of drugs is different in the pediatric population due to several anatomical, biochemical, and physiological differences.(Hines et al. 2002) The full extent of adverse consequences of under- or over-dosing in this population is unknown.(Baber 2003) This lack in knowledge can be attributed to difficulties in studying the pediatric population such as the volume and frequency of blood sampling required, and recruitment of pediatric subjects who are considered to be vulnerable.(Pandolfini et al. 2005) To address these concerns, several countries have introduced laws to improve knowledge of pediatric dosing such as the 2002 Best Pharmaceuticals for Children Act in the United States. (Knibbe et al. 2011)  The lifetime incidence of depression in women is approximately 10-25%. There is a greater vulnerability for the onset or recurrence of depression during the childbearing years between 25 to 44 years old.(Grigoriadis et al. 2007; Kessler et al. 1993) Approximately 5% of pregnant women reporting depression-like symptoms are on antidepressant therapy, such as the selective serotonin reuptake inhibiter (SSRI) class of drugs. One of the most prescribed SSRIs that is also used during pregnancy is fluoxetine  1  (FX). The prevalence of SSRI use during pregnancy results in 5% of all newborns in our population with in utero SSRI exposure.(Bennett et al. 2004; Evans et al. 2001; Oberlander et al. 2006)  The studies in this thesis investigate the ability of the newborn lamb to clear FX from their system, and the development of this ability as newborn lamb age. These studies utilized chronically catheterized lambs and therefore overcome the limitations of performing such studies in humans and smaller animal models. This sheep model builds on previous studies in our group that used pregnant, non-pregnant, and newborn sheep to characterize the maternal, fetal, and neonatal disposition of clinically relevant compounds. Furthermore, this thesis builds on previous research in our group that investigated maternal-fetal fluoxetine disposition in humans and sheep(Kim 2004, 2006)  In the introduction, information on the drug fluoxetine is presented with a focus on its physicochemical properties, pharmacology and disposition. Information on depression in pregnant women and therapies, current research on birth outcomes, lack of knowledge regarding the pharmacokinetics of many drug in the pediatric population, and current research on pediatric pharmacokinetics are also presented. Lastly, a rationale of this thesis research, the animal model used, and the research objectives and hypotheses of the studies in this thesis are presented.  2  1.1.1 Physicochemical	
  properties	
   The chemical nomenclature for fluoxetine (FX) is (±)-N-methyl-3-phenyl-3-[(α,α,αtrifluoro-ρ-tolyl)oxy]propylamine. FX has a tertiary carbon bonded to four unique substituents, giving rise to R and S enantiomers – the 3-dimensional chemical structures of these compounds are illustrated in Figure 1.1 as mirror images of each other..  Figure 1.1 The 3-dimensional chemical structures of fluoxetine enantiomers presented as mirror images of each other.  FX is a small, basic, and lipophilic molecule.(Gram 1994) FX has a relatively low molecular weight of 309.33 daltons, and is weakly basic with a pKa of 9.97 due to its amine functional group. FX is highly lipophilic, with an octanol/water partition coefficient of 4.05.(Lemberger et al. 1985)  1.1.2 Pharmacology	
  and	
  therapeutic	
  uses	
    FX is an antidepressant belonging to the selective serotonin reuptake inhibitor (SSRI) family of drugs. (Levine et al. 1987) One of the neuropharmacology theories for depression is the Serotonin Hypothesis.(Vaswani et al. 2003) Serotonin is a  3  neurotransmitter, which is a chemical that acts as a signal between neurons. Neurotransmitters are specific for the receptors they bind and subsequently the responses they elicit. Serotonin has been hypothesized to be involved in appetite, emotion, mood, and sleep. The Serotonin Hypothesis postulates that there is a deficit in the level of serotonin in individuals suffering from depression. SSRIs inhibit the serotonin reuptake transporter, thereby increasing the amount of serotonin available in the synaptic cleft and subsequent serotonin response.(Owens et al. 1994) SSRIs have high affinity for serotonin reuptake receptors and low affinity for other neuroreceptors such as histamine, acetylcholine, and adrenergic receptors; SSRIs are considerably safer with fewer side effects than other classes of antidepressants.(Gourion et al. 2004; Wong et al. 1990)  FX is administered as a racemic mixture of R and S enantiomers.(Baker et al. 2002) FX was initially approved by the FDA in 1987 for the treatment of depression, and has since been prescribed to over 40 million patients; FX’s prominence was evident when it was featured on the cover of the March 1990 issue of Newsweek as “a breakthrough drug for depression”, and was listed as one of the “Pharmaceutical Products of the Century” in the November 1999 issue of Fortune.(D. Wong et al. 2005) FX is also one of a few antidepressants that is approved for use in children and adolescents (Ludwig et al. 2009), and is increasingly used during pregnancy(Cooper et al. 2007). Despite having been on the market for over four decades, FX continues to be one of the most prescribed antidepressants worldwide.(Ludwig et al.2009) Currently, FX is FDA approved for the treatment of major depression, obsessive-compulsive disorder, bulimia nervosa, panic disorder, and premenstrual dysphoric disorder. Further, FX is FDA approved in  4  combination with olanzapine for the acute treatment of depressive episodes associated with bipolar I disorder, and for adult patients with treatment resistant depression.(Israili et al. 2001; US FDA 2010) 1.1.3 Pharmacokinetics	
  ADME	
   1.1.3.1 Adsorption	
    FX is well absorbed after oral administration. Bioavailability is ~72-90% and peak plasma concentrations occur between 6 to 8 hours. Co-administration with food does not have an effect on the extent of absorption but delays the time to reach peak plasma concentration by 3 to 4 hours.(Lemberger et al. 1985; Altamura 1994; Benfield 1986) 1.1.3.2 Distribution	
   In blood, many drugs are bound to plasma proteins in a reversible manner which may be considered to obey the law of mass action; an equation of this relationship is shown in Figure 1.2 (M. Wood 1986)  Figure 1.2 A proposed kinetic equation describing the mass transfer process of drug to plasma protein binding.  In addition to plasma proteins, many drugs are also bound to other blood constituents, such as red blood cells. The degree of protein binding has important pharmacokinetic and  5  pharmacodynamic implications. The unbound moiety that readily diffuses across biological membranes, reaches receptor sites, and produces pharmacologic effect is also the most readily available form for elimination from the body.(M. Wood 1986)  FX is extensively distributed in the body. Studies in humans found FX to have a large apparent volume of distribution ranging from 14-100 L/kg.(Bergstrom et al. 1988) In adult sheep, the volume of distribution was at the lower end of the human range at 15.1 L/kg.(Kim 2004) FX is highly protein bound (> 90%) in humans and sheep. (Kim 2004; Aronoff et al. 1984)  The major drug binding proteins in blood in humans are albumin, alpha-1-acid glycoprotein (AAG), and lipoproteins. Studies have implicated AAG as the major plasma protein to which FX binds.(Holladay et al.1998) In general, the affinity of a drug towards blood protein is based on the pka of the drug: acidic drugs have affinity toward albumin; and basic drugs have affinity toward AAG. (Wilting et al. 1979; Sager, et al. 1979; Hinderling et al. 2005; Vallner 1977) However, the binding of a drug to a specific blood protein is not believed to be exclusive. (Hervé et al. 1996; Urien et al. 1993; Müller 1985) Rather, depending on the specific physicochemical properties of a drug, it may bind to varying extent to different blood proteins. Although no studies had quantified the extent of FX binding to the specific blood proteins, it may be inferred from studies of similar drugs in the literature that FX has high affinity towards AAG, but also binds to albumin  6  and lipoproteins. (Israili et al. 2001; Otagiri et al. 1989; Eap et al. 2001; M. L. Friedman et al. 1985; Lockwood et al. 1983; De Leve et al. 1981; Schmid et al. 1976)  Further FX undergoes lysosomal trapping, mainly in the lungs which are enriched with lysosomes.(Pohland et al. 1989) FX circulates in the blood in a non-ionized state, and readily permeates cellular membranes. When FX permeates into the acidic interior of lysosomes, it is protonated and become trapped as it is no longer able to permeate the membrane in a charged state.(Daniel et al. 1997) Studies suggest the level of FX in the brain exceeds that in plasma by several fold. (Renshaw et al. 1992; Pohland et al. 1989; Fuller et al. 1993)  1.1.3.3 Metabolism	
    FX is extensively metabolized in the liver. A proposed metabolic scheme is shown in Figure 1.2.(Altamura et al. 1994) FX is mainly metabolized in an Ndemethylation reaction to norfluoxetine (NFX) by CYP2D6, and to a lesser extent CYP2C9, CYP2C19 and CYP3A4. (Kim 2004; Margolis et al. 2000; Fjordside et al. 1999) Clinical pharmacokinetic studies implicated a pivotal role for CYP2D6 in the biotransformation of FX when poor metabolizers were compared with extensive metabolizers. CYP2D6 poor metabolizers had a 2-3 fold greater exposure to FX than CYP2D6 extensive metabolizers. (Hamelin et al. 1996; Fjordside et al. 1999) Several in vitro studies have implicated multiple CYPs in the biotransformation of FX, although the identity and contribution of these CYPs are inconsistent. Reaction phenotyping studies  7  using human liver microsomes and scaled enzyme activity (to reflect variable enzyme levels) implicated CYP2D6, CYP2C9, and CYP3A4 in the metabolism of FX. (Margolis et al. 2000; Ring et al. 2001) In a study using cDNA-expressed CYP isozymes and therapeutic levels of FX implicated CYP2D6, CYP2C9, CYP2C18, and CYP2C19, and no activity was observed with CYP3A4. (Kim 2004)  The pharmacokinetics of FX has been reported to be non-linear. Initial studies found disproportionately higher plasma concentrations with increasing doses. (Altamura, Moro, and Percudani 1994; Bergstrom et al. 1988) FX is an inhibitor of CYP2D6 by mechanism-based inactivation. FX is oxidized by CYP2D6 to a reactive metabolite that binds covalently to the apoprotein or heme group of the CYP enzyme and results in permanent inactivation of the CYP. In order to overcome this inhibition, new CYP enzymes need to be synthesized.(Bertelsen et al. 2003) Mechanism-based inactivation of CYP2D6 by FX is believed to be the mechanism for nonlinear PK.  8  Figure 1.3 A schematic diagram of fluoxetine’s proposed Phase I and II metabolic pathways.  The O-dealkylation reaction of fluoxetine, norfluoxetine, and fluoxetinecarboxylic-acid to trifluoromethylphenol (TFMP) is suggested to be mediated by CYP2C19 and CYP3A4.(Liu et al. 2002) TFMP levels have been quantified in human and rodent studies, but not in sheep.(Urichuk et al. 1997)  Recently, dissertation work in our group investigated hepatic mRNA and enzyme nucleotide and amino acid sequences of several phase I and II orthologues in fetal, 9  newborn, and adult sheep. (Pretheeban et al. 2012; Pretheeban et al. 2011) Compared to humans, the sheep CYP2C19 orthologue had 77% nucleotide and 67% amino acid sequence homology. The sheep CYP2D6 orthologue nucleotide had 80% homology, and amino acid sequence had 76% homology compared to humans. Additionally, sheep HNF4α orthologue nucleotide had 77% homology, and amino acid had 97% homology to human.(Pretheeban et al. 2012) HNF4α (hepatocyte nuclear factor 4 alpha) is a nuclear transcription factor involved in the regulation of hepatic genes, such as the CYP enzymes. These findings suggest a high homology between the phase I hepatic enzymes involved in the metabolism of FX between sheep and humans.  UDP-glucuronosyltransferases (UGT) are a family of phase II hepatic enzymes responsible for the conjugation reaction of a substrate to a glucuronide. Of all the phase II reactions, glucuronidation is quantitatively most prevalent. Sixteen human UGT protein families have been identified: UGT 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2A1, 2B4, 2B7, 2B10, 2B11, 2B15, 2B17, and 2B28.(Uchaipichat et al. 2004) Literature on the specific UGT isoform involved in the metabolism of FX is scarce. According to Tehply et. al., many UGTs are capable of reacting with more than one xenobiotic and with different classes of xenobiotic. Furthermore, numerous drugs are substrates of more than one isoform of UGT.(Tephly et al. 1990) UGT1A4 has been reported in the literature to be the major UGT involved in the N-glucuronidation of secondary amines, though to a lesser extent UGT1A3 and UGT2B10 have also been implicated.(Bourcier et al. 2010) A study reported that UGT-isozymes involved in the N-glucuronidation of olanzapine (also  10  a secondary amine) were 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, and only 1A4 followed saturation kinetics.(Linnet 2002)  1.1.3.4 	
  Elimination	
    In humans, the elimination half-life of FX is 1-3 days, and 7-15 days for NFX.(Lemberger et al. 1985) In a study using radiolabelled fluoxetine, 65-80% of the administered dose was excreted as parent compound or metabolites in urine, and 12-15% was in the feces.(Lemberger et al. 1985) Of this, 3-10% was recovered as intact parent compound, 7-10% as NFX, 5-7% as FX-glucuronide, 8-10% as norfluoxetineglucuronide, and 20% as hippuric acid. (Lemberger et al. 1985; Bergstrom et al. 1988)  For studies in adult sheep, the elimination half-life of FX was reported to be 17.5 hours, and 37.5 hours for NFX.(Kim 2004) The elimination half-life of FX in sheep is shorter compared to humans, which appears to be the trend with other drugs studied in sheep including etidocaine (Pedersen et al. 2009), lidocaine (Bloedow et al. 1980), metoclopramide (Riggs et al. 1988), and diphenhydramine (Yoo et al. 1993). In these sheep studies, renal excretion of the administered dose as parent compound or metabolites (3.6%) is much less than in humans.(Kim 2004) Furthermore, in previous work on fetal-maternal FX disposition in sheep and humans, metabolism of FX in the fetus was not observed, and fetal FX disposition was largely dependent on maternal routes.(Kim 2004)  11  1.1.4 Stereoselective	
  properties	
  of	
  fluoxetine	
  and	
  norfluoxetine	
   Both FX and its major metabolite NFX have a chiral center. For drugs that have a chiral center and are administered as racemic mixtures, it is often important to monitor the levels of each enantiomer when determining pharmacological and pharmacokinetic properties.(R. Lane et al. 1999) Fluoxetine was found to have stereoselective disposition in both human and sheep. (Kim et al. 2004; Kim et al. 2006) In vitro studies suggest that SFX is six times more potent as an inhibitor of CYP2D6 than RFX.(Wong 1990) As a serotonin inhibitor, SNFX has a potency similar to that of the parent and is 22 times more potent than RNFX (DeVane 1999).  Differential three-dimensional interactions between enantiomers of chiral drugs and binding sites on proteins, such as metabolizing enzymes, plasma proteins, transporters, and receptors have been suggested to be the mechanism for stereoselective disposition.(Baker and Prior 2002) Stereoselective metabolism of FX by CYP2D6 in humans have been reported in the literature.(Stevens et al. 1993) Moreover, stereoselective FX metabolism was demonstrated using cDNA preparations of CYP isozyme microsomes, and stereoselective FX plasma protein binding with levels of SFX greater than RFX in sheep and humans. (Kim 2004; Kim 2006)  12  1.2 Depression	
  and	
  pregnancy	
   1.2.1 Incidence	
  of	
  depression	
  during	
  pregnancy	
   The lifetime incidence of depression in women is approximately 10-25%; there is a greater vulnerability for the onset or recurrence of depression during the childbearing years between 25 to 44 years old. (Grigoriadis et al. 2007; Kessler et al. 1993) Approximately 5 to 15% of pregnant women report depressive symptoms at some point during pregnancy, and approximately 30-35% of these patients are prescribed SSRI therapy. (Bennett et al. 2004; Evans et al. 2001; Oberlander et al. 2006) Anxiety, which is reported in approximately 8% of pregnancies, is the second most prevalent affective disorder during pregnancy. Anxiety symptoms receive less research attention perhaps because symptoms are often associated with those of depression during pregnancy. (Evans et al. 2001; Ross et al. 2006)  The treatment of depression in pregnancy using therapeutic drugs is a difficult decision made by physicians and patients, who must consider the best outcome for both the mother and offspring. (Uchaipichat et al. 2004; Hampton 2006) There are numerous risks associated with untreated depression during pregnancy, making the decision of the physician to forgo the use of therapeutic drugs and/or pregnant women deciding on their own to discontinue treatment a difficult one. (Bonari et al. 2004; Roca et al. 2011; Cohen et al. 2010) The psychopathologic symptoms of untreated depression during pregnancy may have direct/indirect physiological consequences for the developing fetus. (Einarson et al. 2001; Kurki et al. 2000; Roca et al. 2011) Several maternal behaviors are associated  13  with untreated depression that place the fetus at risk: effects on a mother’s functional status and ability to seek prenatal care (Einarson et al. 2001); distortion of cognitive abilities to make rational decisions (Bonari et al. 2004); association with poor attendance at antenatal clinics and malnutrition leading to low birth weight (Evans et al. 2001); higher incidence of smoking, use of alcohol and/or other substances of abuse; less likely to take prenatal vitamins and folic acid (Bonari et al. 2004); and high risk of self-inflicted injuries and irrational/impulsive behaviors (Arck 2001; Sugiura-Ogasawara et al. 2002).  Studies have reported the following birth outcomes of women with untreated prenatal depression: increased uterine artery resistance (Teixeira et al.1999); small head circumference; low APGAR scores, higher incidence of special neonatal care; retarded neonatal growth; preterm delivery, higher incidence of high cortisol levels at birth (Chung et al. 2001); higher incidence of operative deliveries; higher incidence of reports of painful labor requiring higher doses of epidural analgesia; and higher incidence of gestational hypertension and subsequent preeclampsia (Roca et al. 2011).  1.2.2 Antidepressant	
  use	
  during	
  pregnancy	
   The use of antidepressants during pregnancy has been on the increase the past decade, despite the lack of complete knowledge regarding the risks of prenatal exposure to such therapies. (Bakker et al. 2008) Because SSRIs have a relatively low incidence of side effects compared to other classes of antidepressants, they are widely used as a firstline treatment for depression and other affective disorders in women of childbearing age. (Casper et al. 2011) Of the many SSRIs on the market, fluoxetine, citalopram, and 14  sertraline are the most commonly prescribed in this population. (Roca et al. 2011; Wisner et al. 2009; Hendrick 2003; Andrade et al. 2011; Vaswani et al. 2003; Ververs et al. 2006) As mentioned previously, approximately 5 to 15% of pregnant women report depressive symptoms at some point during pregnancy resulting in approximately 30-35% of those pregnant women on SSRI therapy. This results in 5% of all newborns in our population with in utero SSRI exposure. (Bennett et al. 2004; Evans et al. 2001; Oberlander et al. 2006)  Risks of psychotropic drug use during pregnancy may include teratogenic effects, direct fetal toxicity, and the potential for long-term neurobehavioral ramifications.(Cohen et al. 1998) Currently, the FDA classifies SSRI drugs in either category C or D. Category C denotes that animal studies suggest risks to the fetus. Category D denotes that human studies suggest harm to the fetus. FX is currently listed in category C. The FDA also provides breastfeeding warnings for FX as a small amount of this drug may be excreted into human milk; however, no adverse events have been reported. (Berlin 2005)  FX treatment during pregnancy may cause a uterine environment with continual fetal SSRI exposure. (Ververs 2009) FX readily crosses the placenta to enter fetal circulation and brain where serotonin and its neurons and receptors are present early during development. (Kinney et al. 2009) Amniotic fluid may be taken up by the fetus by swallowing and drug absorption across the intestinal tract. Studies have reported measurable levels of FX in amniotic fluid, and high fetal (umbilical cord at birth) and  15  maternal FX levels. (Kim et al. 2004; Kim et al. 2006; Hendrick 2003; Rampono et al. 2009)  During fetal development, the serotonin system (neurons and receptors) goes through quantitative maturational changes; thus perturbations to fetal serotonin levels caused by SSRI exposure may result in permanent physiological changes in the offspring. (Branchereau et al. 2002; Lucki 1998) The serotonin system interacts with other neurotransmitter systems that regulate autonomic control. (Bairy et al. 2007; Lu 2006) Further, the serotonin system functions in organizing developing neural networks in the brain and spinal cord. (Branchereau et al. 2002) Broadly, such perturbations to the fetal environment leading to physiological changes that may predispose the fetus to late-onset diseases in later life are referred to as the fetal programming hypothesis. (Gluckman 2006)  1.2.3 SSRI	
  use	
  during	
  pregnancy	
  and	
  birth	
  outcomes	
    Early epidemiological studies did not report any major adverse birth outcomes from antenatal FX exposure. ( Pastuszak et al. 1993; Simon et al. 2002) Subsequently, several groups did report a greater incidence of premature birth and low birth weight. (Chambers et al.1996; Simon et al. 2002; Suri et al. 2007; Toh et al. 2009; Wisner et al. 2009; Rahimi et al. 2006) Recent studies in the past 5 to 10 years have reported serious adverse birth outcomes, such as congenital cardiac defects, persistent pulmonary hypertension, and perturbations in neurodevelopment. However, these reports have been  16  contradictory. Roca (Roca et al. 2011) summarizes the possible shortcomings of these studies: sample size (Costei et al. 2002; Lewis et al. 2010; Oberlander 2002); use of retrospective design or database (Cohen et al. 2000; Källén 2008; Reis 2010; Simon 2002; Toh et al. 2009); and lack of information about maternal mood state during pregnancy (Costei et al. 2002; Källén 2004; Kulin 1998; Lund 2009; Murray 2007; Reis 2010). Wogelius (Wogelius et al. 2006) was the first of many authors to report on a higher incidence of congenital cardiac defects with antenatal SSRI exposure. (Bérard et al. 2007; Diav-Citrin et al. 2008; Louik et al. 2007; Merlob et al. 2009; Pedersen et al. 2009) In contrast, other studies report no increased risk of congenital cardiac malformations.(Alwan et al. 2009)  An increased risk of persistent pulmonary hypertension (PPHN) with antenatal SSRI use, especially during late gestation, has been reported. (Chambers 2006; Gluckman 2006) The prevalence of PPHN is two in one thousand. (Lim et al. 2012) Newborns with PPHN are full-term or near-term without other congenital anomalies; these infants present with severe respiratory failure requiring intubation and mechanical ventilation shortly after birth. (Van Marter et al. 1996) PPHN can result from fetal pulmonary hypertension, which can increase right ventricular afterload and cause pulmonary arterial remodeling. (Steinhorn 2010) Following birth, this can also result in right-to-left shunting of blood through fetal channels, particularly a patent ductus arteriosus. The diminished pulmonary blood flow results in hypoxemia, and an increase in morbidity/mortality. (Van Marter et al. 1996; Abman 1999) In a study by Lim et. al., evidence for increased fetal pulmonary artery pressure was reported following prenatal SSRI exposure; this was  17  associated with transient respiratory problems at birth but not PPHN. However, this study suffered from a small sample size in relation to the low incidence of PPHN. (Lim et al. 2012) Possible mechanisms for the development of PPHN that have been proposed in the literature are as follows: the lung acts as a reservoir for antidepressant drugs and substantial accumulation of SSRIs in the lungs has been reported (Suhara et al. 1998; Lemberger et al. 1985); an increase in pulmonary vascular resistance as serotonin has vasoconstrictive properties (McMahon et al. 1993); mitogenic comitogenic effects on pulmonary smooth-muscle cells (Runo et al. 2003; Eddahibi et al. 2002); proliferation of smooth-muscle cells that is characteristic of PPHN due to elevated serotonin levels in the fetal lung; and inhibitory effect of SSRIs on the synthesis of nitric oxide (a vasodilator) that may be involved in the regulation of vascular tone and reactivity both in utero and during postnatal life (Abman 1999). Currently, five studies have reported an association of PPHN with human antenatal SSRI exposure.(Lim et al. 2012) This risk was higher with SSRI exposure during the second half of pregnancy. (Chambers et al. 2006; Källén et al. 2008) However, other studies have failed to find an association between PPHN and antenatal SSRI use. (Wichman et al. 2009; Andrade et al. 2011)  Several studies have investigated infant neurodevelopment after antenatal SSRI exposure with reports of alterations in regulatory processes observed as changes in arousal, rapid eye movement sleep, and pain responses. The results of these studies in pediatrics, however, have been variable. (Laine et al. 2003; Oberlander et al. 2002; Zeskind et al. 2004) Long-term studies on children with antenatal SSRI exposure have also been variable with some reporting normal mental and psychomotor development and  18  no major effects on cognition, temperament or internalizing and externalizing behaviors (Misri et al. 2006; Nulman et al. 1997; Oberlander et al. 2005), and other studies that reported a higher incidence of a slight delay in psychomotor development. (Casper et al. 2003; Mattson et al. 2004; Mortensen et al. 2003; Pedersen et al. 2010) A recent study suggested an association between antenatal SSRI exposure to an increased incidence of autistic spectrum disorder. (Croen et al. 2011)  The duration of FX and other SSRI use during gestation also appears to be an important factor on birth outcomes with some studies reporting a greater risk for adverse birth outcomes and difficulties with poor neonatal adaptation with longer prenatal exposure. (Oberlander et al. 2008; Casper et al. 2011) Poor neonatal adaptation includes a cluster of symptoms such as restlessness and jitteriness - which are indicative of altered motor activity – and sleep disturbance. (Sanz et al. 2005; Zeskind and Stephens 2004) However, whether these poor neonatal adaptation responses are a result of alterations in neurological development, a withdrawal effect from acute cessation of in utero SSRI exposure at birth, or a combination of both, remains to be elucidated. (Lattimore et al. 2005)  Several studies have examined the effects of prenatal SSRI exposure on delivery outcomes. An increased rate of Cesarean section delivery has been reported for women taking SSRI therapy during pregnancy. (Oberlander et al. 2006; Oberlander et al. 2008; Reis et al. 2010) Studies on the effect of prenatal SSRI therapy on the premature rupture of membranes (PROM) or induction of labor are conflicting, with one group reporting no  19  difference compared to controls while another suggested increased rates. (Pearson et al. 2007; Reis et al. 2010)  1.3 Pediatric	
  drug	
  dosing	
  and	
  disposition	
   1.3.1 Lack	
  of	
  knowledge	
    There is paucity of knowledge on drug disposition in the pediatric population, and proper dosage of many therapeutic drugs for use in this population. The full extent of adverse consequences of under- or over-dosing in this population is unknown. (Baber 2003) To date, only a small number of drugs used in children are licensed for use in this specific subpopulation. (De Cock et al. 2011) A recent study reports that of 140 recently FDA approved drugs that were identified as potentially useful in pediatrics, only 38% were licensed for use in children when initially approved. (Tod et al. 2008) This apparent lack of dosing knowledge has not deterred the use of drugs in this population; the prevalence of off-label drug use in the newborn and infant population has been reported in one study to be 11-37% in community settings, and 55-80% in hospitals (Pandolfini et al. 2005). Another study cites off-label pediatric drug use to be up to 70% in pediatric intensive care, and 90% in neonatal intensive care.(De Cock et al. 2011) Off-label drug use refers to the use of a drug outside its licensed indications in terms of dosage, age, indication, or route of administration.(Conroy 2002)  To address concerns about drug use in this population, several countries have introduced laws to advance knowledge of pediatric dosing. These include the following: 20  the 2002 Best Pharmaceuticals for Children Act in the United States; the 2003 Research Equity Act in Europe; the 2007 Pediatric Regulation Act in Europe; and the FP7 program in Europe that funds research for off-patent drug use in children.(Knibbe et al. 2011)  The lack of specific pediatric information for many drugs can be attributed to difficulties in carrying out clinical trials in this population due to practical and ethical considerations.(Burns 2003) These constraints include the volume and frequency of blood sampling required, and recruitment of pediatric subjects who are often considered to be vulnerable.(Pandolfini et al. 2005) As a result, the off-label dosing of drugs in the pediatric population is often empirically derived from adult regimens which may involve splitting of pills, and dilution of solutions.(Cella et al. 2010)  Several approaches to determining pediatric drug dose have been proposed. The premise for these approaches is based on the use of discrete age points and allometric principles (body weight) that assume linearity in terms of growth from newborn to adults. (Wood et al. 2003; Anderson et al. 2009) However, human growth has been shown to be a nonlinear process; age-related changes in body composition, organ function, weight, and surface area are dynamic, and can be disproportionate especially during the first years of life, which is a period of rapid growth. (Anderson et al. 2000) Such approaches may be acceptable for acute treatment, but risks may arise for continued or long-term treatments. (Wood et al. 2003)  21  1.3.2 Current	
  research	
    There has been great interest recently in the use of population data to derive models to determine optimal drug use in the pediatric population. Databases of drug pharmacokinetics data are collected from published literature and/or sharing amongst clinics, academia, and industry. This conglomeration of data overcomes the limitations of performing empirical pharmacokinetic studies on the pediatric population, and makes use of sparse sampling and uneven data sets that may otherwise provide insufficient information, and reduces the burden on each child. Based on these population datasets, models are derived for optimal use in the pediatric population. (Knibbe et al. 2011; Cella et al. 2010; De Cock et al. 2011)  An example of a population approach is the Nonlinear Mixed Effects Model. Data from all patients are combined and simultaneously analyzed, factoring in variables of the patients from which the data originated (e.g., age, gender, disease state), yielding estimations of PK parameters, and inter- and intra-individual variability. (Knibbe et al. 2011; Meibohm et al. 2005) Such population approach can be considered a “top-down” approach to studying pediatric pharmacokinetics. While these studies hold potential, research in this area is still in its infancy and not without shortcomings. Clinical trials are needed for validation of proposed models.(De Cock et al. 2011) Nedelman (Nedelman 2005) cites concerns for unobserved confounding variables that may bias the inferences of the model, data collection conditions that may cause inaccuracies, correlation analysis of important parameters that may reduce statistical efficiency, and principles of the study  22  design which cannot control costs associated with such studies. In vivo and in vitro studies of pediatric pharmacokinetics are considered as a “bottom-up” approach where the pharmacokinetics of many drugs have been predicted in children (Knibbe et al. 2011), and remain an imperative part of pediatric pharmacokinetics research. (Wood et al. 2003).  1.3.3 Ontogeny	
  of	
  drug	
  disposition	
    The disposition of drugs is different in the pediatric population compared to the adult owing to several factors: gastrointestinal absorptive processes, metabolic ability, level of drug binding blood proteins, expression and activity of drug metabolizing enzymes in the liver and extrahepatic sites, and the geometry of the circulation (ductus venosus and ductus arteriosus). (Besunder et al. 1988; Hill et al. 1988; Rurak et al. 1991; Hines et al. 2002; Tetelbaum et al. 2005) Drugs eliminated primarily by the kidney may be affected by anatomical and functional immaturity of the kidney, low glomerular filtration rate and reduced tubular secretion, and absorptive processes. These differences between the pediatric and adult have been observed in both human and sheep. (H. Wong et al. 2000; van den Anker 1996; Warner 1986; Smith et al. 1989)  Despite inadequate research on pediatric pharmacokinetics, there has been considerable research on the ontogeny of drug metabolizing enzymes. There are qualitative and quantitative differences in drug metabolizing enzymes between the neonate and adult. (Hines et al. 2002; McCarver et al. 2002) The level of drug  23  metabolizing enzymes is low in the fetus and neonate. Studies suggest the overall expression of all isozymes to be 30-50% of adult values. In general, levels increase after birth and reach adult levels within 10 years. (Shimada et al. 1996)  The 3 major cytochrome P450 (CYP450) monooxygenase enzyme families 1 to 3 have variable development between and within each family. (Hines et al. 2002) The levels and activities of several CYP450 isozymes undergo abrupt changes during the perinatal period. CYP450 isozymes relevant to the studies reported in this thesis are those implicated in the metabolism of FX. The activity of CYP2C has been found to be low in the fetal liver and increases rapidly during the first 24 hours following delivery; this rise is due mainly to increased CYP2C levels. (Treluyer et al. 1997; Koukouritaki et al. 2004) Low CYP2D6 levels have been reported in the fetal liver after birth, and to reach adult levels within days to weeks. (Hines et al. 2002; Treluyer et al. 1997) In contrast, CYP3A total protein levels in the early fetus to adult are similar, with levels being 30-40% of total CYP450 in the liver and intestine. (Lacroix et al. 1997; Zajecka et al. 2002) However, the fetus predominantly expresses CP3A7. After birth, there is a progressive switch from the expression of CYP3A7 to 3A4; this switch results in low levels of both CYP3A7 and CYP3A4 during the newborn period. (Stevens et al. 2003)  1.4 Study	
  rationale	
  and	
  experimental	
  model	
    Drug use during pregnancy and for newborns is in general discouraged due to lack of detailed information regarding safety and disposition during these periods. However,  24  the need to use drugs for the treatment of various disorders during these periods is imperative and unavoidable. The prevalence of depression in women of childbearing age results in a sizable portion of pregnant women on SSRI antidepressant therapy, which in turn results in a sizable portion of all newborns in the population with antenatal SSRI exposure. Previous research in our group had identified maternal routes as the major route for FX disposition for the fetus in sheep. (Kim 2004) However, after birth the offspring must rely on his/her own immature mechanisms. The studies in this thesis investigate the ability of newborn sheep to dispose of FX, and the maturation of FX disposition as the newborn ages. This research contributes to the lack of information on the disposition of many drugs in the pediatric population - a thorough understanding of the ontogeny of drug disposition is essential to determine the development of these clearance mechanisms from birth to adult life with the goal of providing clinicians with guidelines for optimal drug dosing.  These studies utilized chronically catheterized lambs from newborn to one year of life. This animal model overcomes the limitations of performing such studies in humans and smaller animal models. Further, sampling frequency and volume of biological fluids that could be collected using chronically catheterized lambs allowed for more detailed studies. Several species differences between humans and smaller animal models such as the rodent would confound the extrapolation of findings to humans: the body mass of fetuses of rodent species, which likely imposes greater metabolic demands upon the mother; rodent species are markedly immature at birth compared to the sheep and humans; and postnatal alternations in physiological functions develop over a much  25  shorter time course compared to humans and sheep(Osmond et al. 1993; Langley-Evans et al. 2005).  The sheep model builds upon previous studies in our group that used pregnant, non-pregnant, and newborn sheep to characterize the maternal, fetal, and neonatal pharmacokinetics and metabolism of clinically relevant compounds such as etidocaine (Pedersen et al. 2009), lidocaine (Bloedow et al. 1980), metoclopramide (Riggs et al. 1988), diphenhydramine (Yoo et al. 1993), fluoxetine (Kim 2004) and valproic acid (H. Wong 2000). Furthermore, the studies in this thesis on postnatal fluoxetine disposition build upon previous fluoxetine research on maternal-fetal pharmacokinetics and metabolism using the sheep animal model. (Kim et al. 2004; Kim et al. 2006) Overall, these studies suggest the fetus had limited ability to metabolize FX, and was dependent on maternal routes to dispose of this drug. The studies in this thesis investigate the ability of newborns to dispose of FX, and the development of disposition abilities up to one year of life.  1.5 Research	
  objectives	
  and	
  hypothesis	
    Using chronically catheterized lambs from a few days after birth up to approximately one year of age and the drug fluoxetine, the following are the research objectives of this study:  •  examine the ability of newborns to eliminate fluoxetine;  26  •  determine the ontogeny of fluoxetine drug disposition ability from a few days following birth up to 1 year of age;  •  determine potential sex differences in the disposition of fluoxetine;  •  assess the stereoselective disposition of fluoxetine over the first few days of life up to 1 year of age.  The hypotheses for the studies in this thesis are as follows: FX disposition in the newborn will be limited compared to the adult due to premature drug disposition mechanisms; though limited, drug disposition in the newborn will be greater than the fetus due to rapid changes in the body during parturition; and as the newborns age, their drug disposition abilities will mature and eventually reach adult levels.  2 Methods	
   2.1 Experimental	
  model	
   All studies were approved by the University of British Columbia Animal Care Committee and Canadian Council on Animal Care. Twenty-two ewes were bred resulting in the birth of 37 lambs (18 females, 19 males) that were used for 71 experimental studies. Experiments were performed at approximately 5, 10 days, 1, 3, 6, and 12 months after birth. The nomenclature used to identify these different age groups is summarized in Table 2.1.  27  Table 2.1 Nomenclature used to classify lamb age groups from newborn to ~1 year of age. Approximate Age Group 5 days 10 days 1 month 3 months 6 months 12 months  Name Cohort #1 Cohort #2 Cohort #3 Cohort #4 Cohort #5 Cohort #6  2.1.1 Breeding	
   Dorset and Suffolk mix-breed ewes were obtained from Three Gates Farm (Gabriola, British Columbia). Their estrous cycles were experimentally synchronized in order to breed lambs available for studies year round in accordance to published methods. (Rhind et al. 1980; Foster 1976; Thorburn 1969; Bassett et al. 1969) In brief, progestin is delivered to the ewes using a vaginal pessary (Pharmplex, Australia) for approximately 14 days, at which time pregnant mare serum gonadotropic (Wyeth, Canada) was injected to induce ovulation. The ewes were then paired with a ram for mating. Conception was confirmed using plasma progesterone levels measured by the Gynecologic Endocrinology Laboratory in the Division of Reproductive Endocrinology and Infertility (UBC, Canada).  Pregnant ewes were transported to the Child and Family Research Institute (Vancouver, British Columbia) at approximately 120 days gestation to allow acclimation to the lab environment prior to delivery at approximately 145 days. Lambs were housed with their mother until the time of weaning at approximately 1 to 2 months of age, at which time the ewes were returned to the farm.  28  2.1.2 Surgical	
  preparation	
   Surgeries to implant catheters in an artery and vein were performed on lambs on postnatal day 1; the catheters were imperative in order to obtain serial blood samples necessary for the experiments. The lamb was anesthetized using 2% isoflurane (Baxter, Illinois, U.S.A) and 40% nitrous oxide (Praxair, Connecticut, U.S.A) delivered using a facemask. When the lamb became unconscious and unresponsive, he/she was intubated with an endotracheal tube (3.5-5.0 mm O.D., Hudson RCI, U.S.A) and anesthesia was maintained using 1-3% isoflurane in laboratory air and oxygen (Praxair, Connecticut, U.S.A) ventilated at a rate adjusted for newborn lamb.(Moss et al. 1995) Eyes were kept lubricated using Refresh (Allergan, California, U.S.A), and analgesia and prophylactic antibiotic were administered using 0.2 mg/kg Meloxicam (Boehringer-Ingelheim, Binger, Germany) and 250 mg ampicillin (Novopharm, Ontario, Canada), respectively. Before the start of the surgery, the skin area around the incision was shaved, sanitized using isopropyl alcohol (Sigma-Aldrich, Milwakee, U.S.A) and povidone-iodine (Dynarex, New York, U.S.A), and locally anesthetized using 0.5% bupivacaine (AnstraZeneca, London, U.K.). A heparin-bonded 3.5 French polyurethane catheter (Intech, PA, U.SA) was inserted into a carotid artery and jugular vein using aseptic surgical technique.  For sanitation purposes, the lambs’ tail was docked.(Hale et al. 2010) The incision area was shaved, disinfected, and locally anesthetized using 0.5% bupivacaine (AstraZeneca, London, U.K.). The tail was cut and cauterized using a Supervet Electric Tail Dock Cutter (Syryet, Iowa, U.S.A). 29  2.1.3 Routine	
  maintenance	
    The health of the lambs was monitored daily using parameters such as weight, appearance of surgical incisions, physical appearance, activity, and food intake. The incision areas were sterilized daily using isopropyl alcohol (Sigma-Aldrich, Milwakee, U.S.A). Catheter patency was maintained daily by flushing with sterile heparinized saline containing 12U of heparin (Organon, Ontario,Canada) per milliliter of 0.9% NaCl solution (Baxter, Illinois, U.S.A).  2.2 Experimental	
  protocols	
   2.2.1 Fluoxetine	
  preparation	
    To prepare 10 mg/ml of sterile racemic fluoxetine, approximately 110 mg of racemic fluoxetine hydrochloride (TRC, Toronto, Canada) was weighed on an analytical balance, dissolved in deionized water, and quantitatively transferred to a 10 ml volumetric flask. This solution was sterilized by filtering through a 0.22 µm nylon syringe filter (Pall, Michigan, U.S.A) into a capped sterile evacuated glass container (Hospira, Illinois, U.S.A). This solution was stored at 4oC until use for up to 3 months.  30  2.2.2 Urine	
  collection	
    For female lambs, a 6 to 14 French Foley urethral catheter (Bard Medical, New Jersey, U.S.A) was inserted under isoflurane anesthesia using aseptic technique the night prior to the start of the pharmacokinetic experiment to minimize the catheter’s occupancy time. For the younger lambs, the identification of the urethra in order to insert the urethral catheter was difficult and insertion of the urethral catheter was not possible for 2 lambs in Cohort #1, 3 lambs in Cohort #2, and 2 lambs in Cohort #3. The catheter was removed from the lamb before the end of the experiment for 2 lambs in each of Cohort #5 and #6.  For male lambs, it was not possible to insert a urethral catheter due to the labyrinth anatomical nature of the male urethra, as shown in Figure 2.1  Figure 2.1 Schematic drawing of the male lamb urethra.  31  A device, inspired by a published method to quantitatively collect urine from cattle, sheep, and swine (Paulson and Cottrell 1984), was designed to collect urine externally as shown in Figure 2.2. A urine collection bag (Bard Medical, New Jersey, U.S.A) that was cut short covered the penis and acted as a funnel connecting to another urine collection bag. The latter collection bag is intact, only allows the one-way entrance of fluid, and has a stop valve where collected urine is drained and stored for subsequent analysis. This set-up was sewn to several straps that were securely tied to the lamb’s abdomen. Sheep void when they stand up(Alexander and Stevens 1982) allowing the urine to be shunted to the collection bag by gravity and securely stored there until sample collection.  Figure 2.2 Urine strap and collection bag arrangement used to collection urine from male lambs. 	
    32  2.2.3 Pharmacokinetic	
  experiments	
    The venous catheter was initially flushed with 1.5 ml heparinized saline to confirm patency. A weight-adjusted IV bolus dose of 1 mg/kg was subsequently injected over approximately 1 minute via the venous catheter. After drug injection, a volume of heparinized saline equal or greater than the dose was injected to flush the drug out of the catheter and into systemic circulation.  Using a 3 ml syringe (BD Biosciences, New Jersey, U.S.A), blood samples were collected from the arterial catheter at -30, -15 minutes before dosing, and 5, 15, 30, 45 minutes, 1, 2, 4, 6, 9, 12, 24, 36, 48, 60 and 72 hours after dosing. Approximately 1.0 to 1.5 ml of blood was collected for the 3 and 10 day old age groups, and 3.0 to 3.5 ml of blood for the older age groups. Prior to blood sampling, the catheter was cleared by withdrawing a volume of heparinized saline and blood equivalent to that to be collected in order to eliminate heparinized saline from the catheter. Upon sample collection, the initially withdrawn blood in heparinized saline was injected back to the lamb, and the catheter was flushed using 3.0 ml of heparinized saline. The collected sample was immediately transferred to a lithium heparin lined Vacutainer tube (BD Biosciences, New Jersey, U.S.A) and mixed thoroughly with the anti-coagulant. The Vacutainer tube was then centrifuged (Beckman Coulter, California, U.S.A) at 2000g for 20 minutes to separate the plasma. The plasma was transferred to a clean screw-top borosilicate test tube (Fisher Scientific, Massachusetts, U.S.A) with a polytetrafluoroethylene lined cap (Fish Scientific, Massachusetts, U.S.A). The samples were stored at -80oC until analysis.  33  Urine was quantitatively collected up to 72 hours after dosing at the following time points: 1, 2, 4, 6, 9, 12, 24, 36, 48, 60, and 72 hours. The volume of urine collected at each interval was measured using a graduated cylinder, and a 10 ml aliquot was collected in a borosilicate test tube with a polytetrafluoroethylene lined cap (Fisher Scientific, Massachusetts, U.S.A) for drug and metabolite measurements. The samples were stored at -80oC until analysis.  2.3 Analytical	
  methods	
   2.3.1 LC/MS/MS	
  assay	
  for	
  plasma	
  samples	
    A sensitive and enantioselective liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed and validated for the simultaneous quantitation of (R)-, (S)- fluoxetine, and (R)-, (S)-norfluoxetine enantiomers in ovine plasma using (R)-, (S)-d5fluoxetine as internal standards.(Chow 2010) This method offers improvements over those previously reported by achieving the following: enantioselectivity and baseline resolution of all enantiomers through the use of a commercially available chiral column; a single-step liquid–liquid extraction using methyl-tert-butyl ether; sensitivity equal to or better than what has been reported; a sample runtime that is comparable if not shorter than what has been reported and a complete analytical method validation of (R)-, (S)-fluoxetine and (R)-, (S)-norfluoxetine.  34  2.3.1.1 Instrumentation	
  and	
  experimental	
  conditions	
    The LC/MS/MS system consisted of a Waters Acquity HPLC Binary Solvent Manager and a Waters Acquity UPLC Sample Manager connected to a Waters Quattro Premier XE triple quadrupole mass spectrometer. The mass spectrometer was operated in electrospray positive ionization (ESI+) mode, and data were acquired using MassLynx v. 4.1 software.  Chromatographic separation was achieved using a micro AGP- CHIRAL column (100 mm × 2.0 mm, 5 µm) connected to an AGP Guard Column (10 mm × 2.0 mm, 5 µm) purchased from Chiral Technologies (West Chester, PA, USA). The column was maintained at ambient temperature (22◦C) and the autosampler tray temperature was maintained at 10◦C to minimize sample evaporation from the 96-well plates. Solvent A was water containing 2 mM ammonium acetate adjusted to pH 4.0 using formic acid, and Solvent B was acetonitrile. The chromatographic conditions were isocratic Solvent A (95%) and Solvent B (5%) with a flow rate of 0.2 ml/min. The total run time was 10 min, and the injection volume was 20 µl.  Mass spectrometric conditions were as follows: capillary voltage 3 kV, cone voltage 30 V, source temperature 120◦C, desolvation gas temperature 400◦C, desolvation gas flow 1000 l/h. Collision Energy (CE) values ranged between 5 eV and 40 eV for the different analytes. Analytes were quantitated using the total ion current (TIC) of the multiple reaction monitoring (MRM) signals of the following transitions, and CE (eV)  35  values: (R)-, (S)-fluoxetine m/z 310.2 → 44.1 (CE 10), 147.7 (CE 20); (R)-, (S)norfluoxetine m/z 296.2 → 30.3 (CE 5), 133.9 (CE 30). (R)-, (S)-d5fluoxetine internal standards were monitored in MRM using m/z 315.2 → 188.1 (CE 5), 122.3 (CE 20) and 152.9 (CE 40). The structural fragments used for MRM analysis are shown in Figure 2.3 Representative ion mass spectra of fluoxetine and norfluoxetine are shown in Figure 2.4. The dwell time was set to 20 ms for (R)-, (S)-fluoxetine and (R)-, (S)-norfluoxetine and 30 ms for internal standard. To protect the mass spectrometer from contamination from the samples and to reduce the solvent load in the source, the mobile phase flow was diverted to the waste prior to 3.5 min and after 8 min during each chromatographic run.  Figure 2.3 Proposed daughter fragments used for multiple reaction monitoring analysis of fluoxetine and norfluoxetine.  36  Figure 2.4 Representative product ion mass spectra (ESI+) of (R)-,(S)-fluoxetine (a), and (R)-,(S)-norfluoxetine (b).  2.3.1.2 Reagent	
  preparation	
    2.3.1.2.1 Preparation	
  of	
  stock	
  solutions	
  and	
  calibration	
  standards	
    A mixed master stock solution consisting of fluoxetine (30 µg/ml), and norfluoxetine (30 µg/ml) was prepared in water. The mixed working stock solution was  37  further diluted with water to yield a series of diluted working stock solutions and used to prepare the calibration standards in ovine plasma. Single-use aliquots of mixed working stock solution were made freshly and stored at -80◦C. Aliquots were thawed and used for each batch to negate any stability issues. The calibration standards were prepared by spiking 50 µl aliquots of appropriately diluted working stock solutions into 250 µl aliquots of blank ovine plasma to yield a final volume of 300 µl. Calibration standards were prepared in the concentration range of 1 to 500 ng/ml for (R)-, (S)-fluoxetine, and (R)-, (S)-norfluoxetine. Each batch of calibration standards was freshly prepared and stored at 4◦C until analysis (not more than 20–30 min). The internal standard (IS) solution was prepared as follows: a master stock solution of d5-fluoxetine (20 µg/ml) was prepared in water, and this was further diluted with water to yield a working stock solution (13 µg/ml); this was stored at 4◦C until analysis. 50 µl of the working stock solution was spiked into all calibration standards, quality control (QC) and plasma samples.  2.3.1.2.2 Preparation	
  of	
  quality	
  control	
  samples	
    The mixed master stock fluoxetine solution (30µg/ml) was diluted in water to working stock solutions that were used to prepare QC-Low (3ng/ml), QC-Mid (40 ng/ml), and QC-High (400 ng/ml) samples in blank ovine plasma. For QC samples used for accuracy determination, 50 µl of the appropriately diluted working stock solution was spiked in 250 µl blank ovine plasma; fresh QC-Low, QC-Mid, and QC-High were prepared for each batch analysis. For QC samples used for precision determination, a  38  volume of 7 ml was prepared for each QC-Low, QC-Mid, and QC-High sample by spiking the appropriate amount of diluted working stock standard in blank ovine plasma in a ratio that was consistent with the spiked standard to plasma ratio used for calibration standards. The QC-Low, QC-Mid, and QC-High samples were dispensed in equal aliquots (approx. 350 µl) and stored at -80◦C until use. A fresh aliquot was thawed, and used for each batch analysis.  2.3.1.2.3 Sample	
  preparation	
    Ovine plasma samples were stored at -80◦C until analysis. On the day of sample analysis, the samples were thawed at room temperature and a 300 µl aliquot was used for parent drug and metabolite measurement. To 300 µl of sample, 50 µl of the diluted working stock solution of the IS (13 µg/ml) was added followed by the addition of 300 µl of 0.1 N NaOH solution. The samples were vortex-mixed again for at least 15 s, and then 3.0 ml of methyl-tert-butyl ether was added. The samples were vortex-mixed for at least 45 s. To separate the organic and aqueous phases, samples were placed in -80◦C freezer for at least 10 min, and then the top organic layer was decanted to a new set of tubes. The organic layer was brought to dryness under nitrogen in a sample evaporator set at 35◦C. The dried residues were reconstituted with 100 µl of 2 mM ammonium acetate (pH 4.0) containing 5% acetonitrile.  39  2.3.1.3 Method	
  validation	
    The method was validated for accuracy, precision, linearity, range, LOQ, selectivity, recovery, dilution integrity, matrix effect and carry-over in ovine plasma sample according to the US Food Drug Administration (FDA), Guidance for Industry: Bioanalytical Method Validation.(FDA 2009) Calibration curves included eight calibration levels prepared freshly on each day of a batch analysis with the matrix blank. QC samples for precision determination were freshly thawed on each day of a batch analysis; for accuracy determination, QC samples were freshly prepared.  2.3.1.3.1 Accuracy	
  and	
  precision	
    The results for accuracy and precision are presented in Tables 2.2 and 2.3. Accuracy was expressed as the %deviation for the QC-Low, QC-Mid and QC-High samples. Intra-day accuracy ranges observed for the analytes were as follows: (R)fluoxetine -11.0% to 9.78%; (S)-fluoxetine -11.7 to 5.76%; (R)-norfluoxetine -11.8 to 12.3%; (R)-norfluoxetine -11.8 to 12.3% and (S)-norfluoxetine -11.8% to 7.87%. Interday accuracy observed ranges for the analytes were as follows: (R)-fluoxetine -8.82% to 3.75%; (S)-fluoxetine -10.8% to 1.46%, (R)-norfluoxetine -7.50% to 0.37%, and (S)norfluoxetine -8.77% to -1.33%.  Precision was expressed as the % relative standard deviation (%RSD) for the QCLow, QC-Mid and QC-High samples. Intra-day precision observed ranges for the  40  analytes were as follows: (R)-fluoxetine 2.31–11.7%; (S)-fluoxetine 1.38–8.31%; (R)norfluoxetine 1.49–10.1% and (S)-norfluoxetine 1.60–16.4%. Inter-day precision observed ranges for the analytes were as follows: (R)-fluoxetine 5.29–11.5%; (S)fluoxetine 3.91-11.1%; (R)-norfluoxetine 4.32–7.67% and (S)-norfluoxetine -8.77% to 1.33%.  The method met the acceptance criteria for accuracy (%Deviation) ±20% for the QC-Low, and ±15% for the QC-Mid and QC-High samples. Acceptance criteria for precision (%RSD) ≤20% for the QC- Low, and ≤15% for the QC-Mid and QC-High samples, were met as well. This indicated the method was accurate and precise over the range of the assay.  Table 2.2 Intra-day and inter-day accuracy and precision results for the validation of fluoxetine enantiomers.  Accuracy (%Dev) QC-Low QC-Mid QC-High  R-fluoxetine S-fluoxetine Intra-Day Inter-Day Intra-Day Inter-Day Accuracy Day 1 Day 2 Day 3 Day 1-3 Day 1 Day 2 Day 3 Day 1-3 (%Dev) (n=6) (n=6) (n=6) (n=18) (n=5) (n=6) (n=6) (n=17) 2.60 9.78 -1.81 3.52 QC-Low -2.11 7.32 -8.06 -0.948 8.68 -1.01 3.59 3.75 QC-Mid 5.76 -4.56 3.17 1.46 -5.29 -10.2 -11.0 -8.82 QC-High 5.76 -10.1 -11.7 -10.8  Intra-Day Inter-Day Intra-Day Inter-Day Precision Precision (%RSD) Day 1 Day 2 Day 3 Day 1-3 %(RSD) Day 1 Day 2 Day 3 Day 1-3 (n=5) (n=5) (n=5) (n=15) (n=5) (n=5) (n=5) (n=15) QC-Low 11.7 QC-Mid 2.80 QC-High 3.99  4.3 2.31 3.13  7.3 1.38 1.58  11.5 5.29 7.16  QC-Low 3.60 QC-Mid 5.56 QC-High 3.32  4.33 2.31 3.13  7.30 1.38 1.58  11.1 4.58 3.91  41  Table 2.3 Intra-day and inter-day accuracy and precision results for the validation of norfluoxetine enantiomers.  Accuracy (%Dev) QC-Low QC-Mid QC-High  R-norfluoxetine S-norfluoxetine Intra-Day Inter-Day Intra-Day Inter-Day Accuracy Day 1 Day 2 Day 3 Day 1-3 (%Dev) Day 1 Day 2 Day 3 Day 1-3 (n=6) (n=6) (n=6) (n=18) (n=6) (n=6) (n=5) (n=17) -9.03 6.37 4.73 -3.67 QC-Low -9.59 7.87 -5.86 -1.33 -1.41 -9.82 12.3 0.367 QC-Mid -1.41 -1.41 -9.82 -5.62 -5.77 -11.78 -4.96 -7.50 QC-High -5.29 -5.77 -11.8 -8.77  Intra-Day Inter-Day Intra-Day Inter-Day Precision Precision (%RSD) Day 1 Day 2 Day 3 Day 1-3 (%RSD) Day 1 Day 2 Day 3 Day 1-3 (n=5) (n=5) (n=5) (n=15) (n=5) (n=5) (n=5) (n=15) QC-Low 10.1 7.09 2.42 7.67 QC-Low 16.4 4.38 4.79 10.1 QC-Mid 5.76 3.20 1.49 4.32 QC-Mid 10.9 7.98 1.57 9.06 QC-High 7.99 3.44 1.62 5.17 QC-High 7.99 4.12 2.96 7.61  2.3.1.3.2 Linearity	
  and	
  range	
    Linearity of the calibration curve was evaluated in seven batches over the course of method validation. The coefficient of determination (mean ± SD, n = 4) for (R)fluoxetine was r2 = 0.997 ± 0.0004, (S)-fluoxetine r2 = 0.996 ± 0.0009, (R)-norfluoxetine r2 = 0.989 ± 0.007 and (S)-norfluoxetine r2 = 0.994 ± 0.002. The observed accuracy ranges (%Deviation) of the calibration curve standards were as follows: (R)-fluoxetine 1.35–6.30%; (S)-fluoxetine 1.29 to 7.87%; (R)-norfluoxetine 2.99 to 11.9% and (S)norfluoxetine -2.07% to -9.79%. The observed precision ranges (%RSD) of the calibration curve standards were as follows: (R)-fluoxetine 1.54 to 7.92%; (S)-fluoxetine 0.69 to 7.11%; (R)-norfluoxetine 2.61 to 13.4% and (S)-norfluoxetine 2.11 to 12.9%. The calibration curve met the acceptance criterion for linearity of r2 ≥ 0.980 using a weighting of 1/X2 . The range of the method was established as 1 ng/ml to 500 ng/ml  42  based on acceptance criteria that the calibration levels are accurate (%Deviation ± 15%) and precise (%RSD ≤ 15%). The results indicated that the calibration curve was linear, accurate, and precise over the range of the assay.  2.3.1.3.3 Limit	
  of	
  quantitation	
  and	
  selectivity	
    LOQ was determined to be 1ng/ml. The signal-to-noise (S/N) ratios (mean ± SD, n = 6) for the analytes were as follows: (R)-fluoxetine 13.0 ± 11.8%; (S)-fluoxetine 12.7 ± 6.71%; (R)-norfluoxetine 7.93 ± 2.39% and (S)-norfluoxetine 18.1 ± 11.7%. Accuracy (%Deviation, n = 6) were as follows: (R)-fluoxetine −9.50% to 15.4%; (S)-fluoxetine −7.70% to 18.0%; (R)-norfluoxetine −8.00% to 11.2% and (S)-norfluoxetine −9.90% to 16.9%. Precision (%RSD, n = 6) was as follows: (R)-fluoxetine 7.17%; (S)-fluoxetine 14.8%; (R)-norfluoxetine 14.0% and (S)-norfluoxetine 16.8%. Results for the determination of LOQ met the acceptance criteria of S/N ≥ 5:1, accuracy (%Deviation) ± 20% and precision %RSD ≤ 20%. The method was accurate and precise at the established LOQ of 1ng/ml of (R)-, (S)-fluoxetine, and (R)-, (S)- norfluoxetine using 300 µl of sample.  The selectivity of the method was investigated in five ovine spiked plasma samples spiked at the LOQ level. Blank ovine plasma samples from six separate animals were also prepared. The selectivity met the acceptance criteria of S/N ≥ 5:1 for (R)-, (S)fluoxetine, and (R)-, (S)-norfluoxetine, and there was no significant interference at the retention times of the analytes when the blank ovine plasma samples were compared to  43  the LOQ samples as shown in Figure 2.5. The results indicated that the method was selective for these analytes.  Figure 2.5 Limit of quantitation and selectivity in ovine plasma of racemic fluoxetine in blank ovine plasma (a), racemic fluoxetine in blank ovine plasma (b), racemic fluoxetine 1 mg/ml standard in ovine plasma (c), and racemic norfluoxetine 1 ng/ml standard in ovine plasma.  2.3.1.3.4 Recovery	
    The mean %Recovery values (n = 6) of analytes for QC-Low, QCMid and QC-High were as follows: (R)-fluoxetine 121%, 115% and 114%, respectively; (S)-fluoxetine 122%, 108% and 107%, respectively; (R)-norfluoxetine 75.9%, 99.6%,  44  and 105%, respectively and (S)-norfluoxetine 100%, 96.7%, and 99.5%, respectively. The results indicated that (R)-, (S)-fluoxetine, and (R)-, (S)-norfluoxetine were extracted efficiently over the concentration range of the assay.  2.3.1.3.5 Matrix	
  effect	
    The %Matrix Effect obtained for QC-Low, QC-Mid, and QC-High were as follows: (R)-fluoxetine 7.21%, -3.09%, -4.83%, respectively; (S)-fluoxetine 13.1%, 4.72%, and -0.659%, respectively; (R)-norfluoxetine -7.33%, 1.33% and -12.2%, respectively, and (S)-norfluoxetine 7.96%, 7.54%, and -4.81%, respectively. The results indicated that matrix effect was negligible for this method.  2.3.1.3.6 Dilution	
  integrity	
  and	
  evaluation	
  of	
  carry-­‐over	
    For the pharmacokinetic studies, all ovine plasma samples analyzed were within the concentration range of the method and did not require dilution. However, should the possibility of higher analyte concentrations be encountered for unforeseen studies, the method was validated for dilution integrity. After the 10-fold dilution of the six aliquots of the QC-High samples, the observed accuracy ranges (%Deviation, n=6) were as follows: (R)-fluoxetine -17.8% to -10.0%, (S)-fluoxetine -19.8% to -8.95%, (R)norfluoxetine -19.7% to 19.5%, and (S)-norfluoxetine -19.1% to 9.62%. The precision (%RSD, n = 6) obtained were as follows: (R)-fluoxetine 3.27%, (S)-fluoxetine 6.00%, (R)-norfluoxetine 14.6%, and (S)-norfluoxetine 14.7%. The results met the acceptance 45  criteria of accuracy (%Deviation) ± 20% from the actual value (40 ng/ml), and precision (%RSD) ≤ 15%.  This indicated QC high samples could be diluted 10-fold without losing accuracy or precision. Carry-over between injections of the QC-Mid and blank plasma samples for the analytes and internal standards were as follows: (R)-fluoxetine 0.730%; (S)fluoxetine 0.810%; (R)-norfluoxetine 0.100%; (S)-norfluoxetine 0.430%; (R)d5fluoxetine 0.532%, and (S)- 5fluoxetine 0.756%. All analytes and internal standards met the acceptance criteria of ≤5%.  2.3.2 LC/MS/MS	
  assay	
  for	
  urine	
  samples	
    Urine fluoxetine and its metabolites were quantified by modifying the plasma LC/MS/MS assay.(Chow 2012) The same chiral chromatography system was used. In addition to R,S-fluoxetine, and R,S-norfluoxetine, TFMP was also quantified using a structurally similar compound, 2-Chloro-3-(trifluoromethyl)phenol, as an internal standard.  2.3.2.1 Instrumentation	
  and	
  experimental	
  procedures	
    The LC/MS/MS system consisted of an Agilent 1290 Infinity LC System connected to an AB Sciex QTRAP 5500 triple quadrupole and linear ion trap mass spectrometer. The mass spectrometer was operated simultaneously in electrospray 46  positive (ESI+) and negative ionization (ESI-) modes, and data were acquired using an Analyst v. 1.4 software to allow simultaneous quantitation of R,S-fluoxetine and R,Snorfluoxetine which are ionized to positive ions, and TFMP which is ionized to negative ions.  Chromatographic separation was achieved using a micro AGP- CHIRAL column (100 mm × 2.0 mm, 5 µm) connected to an AGP Guard Column (10 mm × 2.0 mm, 5 µm) purchased from Chiral Technologies (West Chester, PA, USA). The column was cooled to 12◦C to achieve chromatographic separation of analyte peaks and the autosampler tray temperature was maintained at 10◦C to minimize sample evaporation from the 96-well plates. Solvent A was HPLC water containing 2 mM ammonium acetate adjusted to pH 4.0 using formic acid, and Solvent B was acetonitrile. The chromatographic conditions were isocratic Solvent A (95%) and Solvent B (5%) with a flow rate of 0.2 ml/min. The total run time was 20 min, and the injection volume was 20 µl.  Mass spectrometric conditions were as follows: capillary voltage 3 kV, cone voltage 30 V, source temperature 250◦C, desolvation gas temperature 400◦C, desolvation gas flow 1000 l/h. Collision Energy (CE) values ranged between 5 eV and 45 eV for the different analytes. Analytes were quantitated using the total ion current (TIC) of the multiple reaction monitoring (MRM) signals of the following transitions, and CE (eV) values: (R)-, (S)-fluoxetine m/z 310.2 → 44.1 (CE 10), 147.7 (CE 20); (R)-, (S)-  47  norfluoxetine m/z 296.2 → 30.3 (CE 5), 133.9 (CE 30), and para-tri(fluoromethyl)phenol m/z 161.2 → 120.9 (CE -45), 92.9 (CE -35), 73 (CE -20). (R)-, (S)-d5fluoxetine and 2-chloro-3-(trifluoromethyl)phenol internal standards were monitored in MRM using m/z 315.2 → 188.1 (CE 5), 122.3 (CE 20) and 152.9 (CE 40), and 194.9 → 140.9 (CE -22.6), 121.1 (CE -32), and 93.1 (CE -37), respectively. The dwell time was set to 20 ms for all analytes. To protect the mass spectrometer from sample contamination and to reduce the solvent load in the source, the mobile phase flow was diverted to the waste when analyte peaks were note eluting before 3.0 min, between 6 to 9 min, and after 19 min during each chromatographic run. The proposed fragments used for MRM analysis of TFMP is shown in Figure 2.6.  120.9 m/z F F  92.9 m/z  F  O  73.1 m/z  Figure 2.6 Proposed fragmentation ions used for multiple reaction monitoring analysis of TFMP. 2.3.2.2 Reagent	
  preparation	
   2.3.2.2.1 Preparation	
  of	
  stock	
  solutions	
  and	
  calibration	
  standards	
    A mixed master stock solution consisting of fluoxetine (30 µg/ml), norfluoxetine (30 µg/ml), and (30 µg/ml) TFMP was prepared in water. The mixed working stock  48  solution was further diluted with water to yield a series of diluted working stock solutions and used to prepare the calibration standards in ovine plasma. Single use aliquots of mixed working stock solution were made freshly and stored at -80◦C. Aliquots were thawed and used for each batch to negate any stability issues. The calibration standards were prepared by spiking 50 µl aliquots of appropriately diluted working stock solutions into 250 µl aliquots of blank ovine plasma to yield a final volume of 300 µl. Calibration standards were prepared in the concentration range of 1 to 500 ng/ml for racemic fluoxetine, norfluoxetine and TFMP. Each batch of calibration standards were freshly prepared and stored at 4◦C until the analysis (not more than 20-30 min). The internal standard (IS) solution was prepared as follows: a master stock solution of d5-fluoxetine (20 µg/ml) and (10 µg/ml) TFMP-IS was prepared in water, and this was further diluted with water to yield a working stock solution (13 µg/ml d5-fluoxetine, 6.5 µg/ml for TFMP-IS); this was stored at 4◦C until analysis. A 100 µl of the working stock solution was spiked into all calibration standard, quality control (QC) and plasma samples.  2.3.2.2.2 Preparation	
  of	
  quality	
  control	
  samples	
   The mixed master stock of fluoxetine, norfluoxetine, and TFMP solution (30µg/ml) was diluted in water to working stock solutions that was used to prepare QCLow (3ng/ml), QC-Mid (40 ng/ml), and QC-High (400 ng/ml) samples in blank ovine plasma. For QC samples used for accuracy determination, 50 µl of the appropriately diluted working stock solution was spiked in 250 µl blank ovine plasma; fresh QC-Low, QC-Mid, and QC-High were prepared for each batch analysis. For QC samples used for precision determination, a volume of 7 ml was prepared for each QC-Low, QC-Mid, and  49  QC-High samples by spiking the appropriate amount of diluted working stock standard in blank ovine plasma in a ratio that was consistent with the spiked standard to plasma ratio used for the calibration standards. The QC-Low, QC-Mid, and QC-High samples were dispensed in equal aliquots (approx. 350 µl) and stored at -80◦C until use. A fresh aliquot was thawed, and used for each batch analysis.  2.3.2.2.3 Sample	
  preparation	
    The assay had a simple sample preparation procedure. The assay used 200 ul of samples which was diluted 5-fold to 1000 ul with deionized water; a 100 ul aliquot was then combined with another 100 ul of internal standards (R,S-d5fluoxetine, 2-Chloro-3(trifluoromethyl)phenol), then the sample was analyzed by LC/MS/MS. The final dilution was 10-folds.  Preparing the samples by dilution, as opposed to liquid-liquid extraction, simplified subsequent data analysis. The amount of drug/metabolites excreted during any collection interval was calculated as follows: the calculated concentration by LC/MS/MS was multiplied by the 10-folds dilution, and finally by the total volume of urine collected during that interval. Alternatively, had the sample preparation required a liquid-liquid extraction of the analytes out of the urine sample, potential confounding factors such as %recovery from the extraction could have complicated and confounded the data analysis.  50  2.3.2.3 Method	
  validation	
    The assay was validated for the quantitation of R,S-fluoxetine, and it’s metabolites TFMP and R,S-norfluoxetine according to FDA guidelines. (FDA 2009) The assay was validated for accuracy, precision, linearity, range, LOQ, selectivity, dilution integrity, matrix effect and carry-over. Validation results are summarized in Table 2.4 to 2.8. Chromatograms of MRM’s used for the quantitation of TFMP, TFMP-IS, FX, NFX, and d5-FX are shown in Figure 2.7. LOQ and selectivity chromatograms of blank ovine urine and a 1 ng/ml standard in ovine urine are shown in Figure 2.8.  Table 2.4 Intra-day and inter-day accuracy and precision results for the validation of fluoxetine enantiomers  Accuracy (%Dev) QC-Low QC-Mid QC-High  R-fluoxetine S-fluoxetine Intra-Day Inter-Day Intra-Day Inter-Day Accuracy Day 1 Day 2 Day 3 Day 1-3 Day 1 Day 2 Day 3 Day 1-3 (%Dev) (n=6) (n=5) (n=6) (n=17) (n=6) (n=6) (n=5) (n=17) -3.88 -10.02 -10.71 8.20 QC-Low -5.38 -5.50 2.20 3.62 1.63 -2.09 -1.09 4.26 QC-Mid -3.43 03.81 -2.05 3.99 5.89 12.04 2.37 7.11 QC-High 10.87 1.85 7.40 6.76  Intra-Day Inter-Day Intra-Day Inter-Day Precision Precision Day 1 Day 2 Day 3 Day 1-3 Day 1 Day 2 Day 3 Day 1-3 (%RSD) %(RSD) (n=5) (n=6) (n=6) (n=17) (n=6) (n=5) (n=5) (n=16) QC-Low 13.07 12.39 4.85 QC-Mid 7.16 4.06 4.05 QC-High 4.38 13.15 1.23  10.16 5.33 6.95  QC-Low 7.27 QC-Mid 4.63 QC-High 5.09  7.14 10.90 1.60 1.64 8.16 6.90  8.71 3.15 5.82  51  Table 2.5 Intra-day and inter-day accuracy and precision results for the validation of norfluoxetine enantiomers. R-norfluoxetine S-norfluoxetine Intra-Day Inter-Day Intra-Day Inter-Day Accuracy Accuracy (%Dev) Day 1 Day 2 Day 3 Day 1-3 (%Dev) Day 1 Day 2 Day 3 Day 1-3 (n=6) (n=6) (n=6) (n=18) (n=6) (n=6) (n=5) (n=17) QC-Low -11.02 3.29 8.54 7.62 QC-Low 2.03 11.80 15.78 9.87 QC-Mid -4.91 -2.51 6.92 4.78 QC-Mid 8.32 4.85 5.65 7.45 QC-High 13.32 -0.93 5.24 5.87 QC-High 11.30 8.11 11.42 10.41 Intra-Day Inter-Day Intra-Day Inter-Day Precision Precision (%RSD) Day 1 Day 2 Day 3 Day 1-3 (%RSD) Day 1 Day 2 Day 3 Day 1-3 (n=5) (n=6) (n=6) (n=17) (n=6) (n=6) (n=6) (n=18) QC-Low 11.32 10.06 9.84 8.59 QC-Low 10.23 7.30 11.51 9.09 QC-Mid 2.16 1.86 2.11 2.32 QC-Mid 11.41 2.29 4.91 3.71 QC-High 1.39 8.91 2.53 6.82 QC-High 6.91 4.12 4.62 5.39 Table 2.6 Intra-day and inter-day accuracy and precision results for the validation of TFMP. TFMP Intra-Day Inter-Day Accuracy (%Dev) Day 1 Day 2 Day 3 Day 1-3 (n=6) (n=5) (n=6) (n=17) QC-Low 5.48 8.41 5.72 6.54 QC-Mid 5.61 1.32 1.83 2.92 QC-High 3.38 5.44 6.73 6.94 Precision (%RSD) QC-Low QC-Mid QC-High  Intra-Day Inter-Day Day 1 Day 2 Day 3 Day 1-3 (n=5) (n=5) (n=5) (n=15) 2.79 9.77 8.95 7.51 4.05 6.55 7.09 5.70 6.60 2.91 7.02 9.82  Table 2.7 Urine LC/MS/MS assay matrix effect validation results. n=3 QC-Low QC-Mid QC-High  TFMP 3.24% 0.472% 1.47%  RFX 5.23% 1.23% 5.37%  SFX 9.54% 5.33% 4.24%  RNFX 2.64% 4.32% 8.43%  SNFX 5.32% 2.64% 1.42%  52  Table 2.8 Urine LC/MS/MS assay dilution integrity and %carry-over validation results. n=6  TFMP RFX SFX RNFX SNFX %Dev %RSD %Dev %RSD %Dev %RSD %Dev %RSD %DEV %RSD Dilution Integrity 16.2 5.57 5.34 8.51 -3.06 5.35 14.6 1.75 -15.2 3.02 % Carry-Over 0.167% 0.080% 0.201% 0.681% 0.345%  Figure 2.7 Ion mass spectra for the multiple reaction monitoring analysis of TFMP (a), TFMP internal standard (b), fluoxetine enantiomers (c), norfluoxetine enantiomers (d), and d5-fluoxetine enantiomers.  53  Figure 2.8 Limit of quantitation and selectivity in ovine urine blank (a), and 1 ng/ml standard in ovine urine (b).  2.3.2.4 LC/MS/MS	
  analysis	
  of	
  glucuronide	
  conjugates	
  in	
  urine	
    Glucuronide conjugate metabolites were also quantified in the urine samples. The urine samples were incubated with β-Glucuronidase (SigmaAldrich, Missouri, U.S.A) that hydrolyzed the glucuronide to drug/metabolite bond, and the resulting increase in drug/metabolite was measured using the urine assay. The hydrolysis reaction of βGlucuronidase is shown in Figure 2.9.  Figure 2.9 A schematic equation showing the hydrolysis reaction of β-Glucuronidase  54  Since the urine assay was validated to quantitate R,S-fluoxetine, R,Snorfluoxetine, and TFMP, and theoretically these drugs/metabolites are able to form glucuronide conjugates, the increase in R,S-fluoxetine, R,S-norfluoxetine, and TFMP concentrations after enzyme incubation provided an indirect assay for their corresponding glucuronide conjugates. This incubation procedure was described by Dr. John Kim in his dissertation work that quantified fluoxetine and norfluoxetine glucuronide in adult pregnant and non-pregnant ewes and fetal lambs. (Kim 2000) A 200 ul of urine sample was diluted 5-fold in 1000 ul of β-glucuronidase in 0.2M sodium acetate (pH 5) buffer at 37oC for 4 hours; a 100 ul aliquot was then combined with another 100 ul of internal standards (R,S-d5fluoxetine, 2-Chloro-3-(trifluoromethyl)phenol), then the sample was analyzed by LC/MS/MS. This dilution scheme is analogous to the one used for urine R,Sfluoxetine, R,S-norfluoxetine and TFMP analysis, with the exception that for the initial 5folds dilution in water, this was done in β-glucuronidase buffer. This analogy in sample preparation simplifies the calculation of the glucuronide conjugates since the dilution scheme was the same.  2.3.3 Plasma	
  protein	
  binding	
    Plasma protein binding of FX and NFX was determined in vivo and ex vivo, respectively, using equilibrium dialysis procedure described and validated by Dr. John Kim.(Kim 2000) Equilibrium dialysis is one of the most commonly used methods for the study of drug plasma protein binding. (Routledge 1986; M. Wood 1986; Piafsky and Woolner 1982; De Leve and Piafsky 1981)  55  The plasma sample was allowed to equilibrate with a protein-free phosphate buffer solution. The plasma and buffer compartments were separated by a semipermeable dialysis membrane consisting of pores that exclude the passage of plasma proteins. Thus, unbound drug was allowed to pass freely through the membrane and equilibrate between the buffer and plasma fractions. The fraction of fluoxetine bound to plasma protein was calculated using the following equation: (Barre et al. 1985; Tozer et al. 1983)  PC = Test compound concentration in plasma protein containing compartment PF = Test compound concentration in plasma protein free buffer compartment. fu = fraction of unbound drug.  Equilibrium dialysis was carried out at 39oC using 1ml Plexiglass® dialysis cells and cellophane dialysis membranes with a molecular weight cutoff of 10 kDa (SigmaAldrich, U.S.A). The dialysis membrane was rinsed with deionized water and soaked in isotonic phosphate buffer for a minimum of 1 hour at 39oC prior to use. Aliquots (500 µL) of plasma sample were dialyzed against an equal volume of pH isotonic phosphate buffered saline (Invitrogen, U.S.A). The equilibration time was 4 hours after which the plasma and buffer were assayed for FX and NFX using the previously described LC/MS/MS assay.  56  Dissertation work by Dr. John Kim identified two methods to study drug protein binding.(Kim 2000) The ex vivo method measures protein binding of the drug already in the collected plasma sample. The drug was administered to the experimental model and protein binding was measured in the collected plasma. This method presents the advantage of measuring protein binding most similar to physiological conditions. However, a caveat of this method is that the level of the drug must be sufficiently high enough to measure protein binding, which was calculated to be 20 ng/ml. A diagram of the mechanism of protein binding, and calculation of minimum drug requirement is presented in Figure 2.10.  Figure 2.10 Schematic diagram of the mechanism of equilibrium dialysis. A drug level of 20 ng/ml for FX and NFX was not consistently observed for the samples used for protein binding analysis, especially for the younger cohorts. Therefore, protein binding was determined using the in vivo method.  57  Fluoxetine and norfluoxetine protein binding was determined in vivo by spiking the pre-dose control samples with ~100 ng/mL of fluoxetine and norfluoxetine and incubating for 1 hour at 39oC; these incubation conditions and stability studies were optimized by Dr. John Kim. (Kim 2000) Equilibrium dialysis experiments were then performed on the incubated samples.  2.3.4 Alpha-­‐1	
  Acid	
  Glycoprotein	
  immunoassay	
    The level of AAG in plasma samples was quantified using a commercially available immunoassay (Quantikine Inc., U.S.A) The in vivo samples used for equilibrium dialysis studies were used – these were control samples collected pre-dose during pharmacokinetic studies, and were pooled by age and sex. Sample preparation and assay protocols were followed exactly with the exception that samples were initially diluted 300-fold.  2.3.5 Bradford	
  immunoassay	
    The total protein level in plasma samples was quantified using a commercially available immunoassay (Millipore, Massachusetts, U.S.A).The in vivo samples used for the equilibrium dialysis studies were used – these were control samples collected predose during pharmacokinetic studies, and were pooled by age and sex.  58  2.3.6 Blood	
  pH	
  and	
  temperature	
    Blood acid-base balance was measured using an I-STAT-1 analyzer (Abbott, Ontario, Canada) with CG8+ cartridges (Abaxis, California, USA). Body temperature was measured using a thermistor rectal probe (YSI Instruments, Ohio, USA).  2.4 Pharmacokinetic	
  analyses	
    Pharmacokinetic parameters were estimated by standard pharmacokinetic procedures in accordance with Gibaldi and Perrier.(Perrier and Gibaldi 1982) Data modeling (compartmental and non-compartmental) was assisted by the use of Phoenix WinNonLin (Pharsight, California, U.S.A). Parameters estimated in plasma include area under the curve (AUC), total body clearance (ClTB), volume of distribution at steady state (Vdss), mean residence time (MRT), elimination half-life (t1/2). Parameters estimated in urine include amount accumulated (Xu0 -∞), renal clearance (Clr), and dose recovered in urine (%dose).  The concentration versus time values were modeled using a weighting factor of 1/y2. The goodness of fit of a model to the data set was evaluated using Akaike’s Information Criterion (AIC) and Schwarz Criterion (SC). The model that minimized AIC and SC values was regarded as having the best fit to the data set. The elimination rate constant (β) was estimated by determining the terminal slope of the best fit model.  59  The area under the plasma concentration-time curves (AUC0 -∞) was estimated by the summation of (AUC0 -tlast) and (AUC tlast -∞). (AUC0 -tlast) was estimated using the linear trapezoidal rule, and (AUC tlast -∞) was estimated by dividing the concentration of the last time point sampled by the β.  Total body clearance was calculated by the following equation:  The volume of distribution was estimated using non-compartmental analysis. By using the first and zero moment curves, the variability of curve fitting by compartmental analysis is minimized. The formula used was as follows:  Mean Residence time was estimated using non-compartmental analysis by dividing the area under the first moment curve by the area under the zero moment curve according the following equation:  Elimination half-life was calculated by the following equation:  60  2.5 Statistical	
  analysis	
    Two-way ANOVA with age and sex as the dependent variables, and post-hoc Tukey-Kramer’s analysis were used for plasma and urine PK parameters, plasma protein binding, AAG and Bradford Immunoassays, and blood pH and temperature studies. To analyze stereoselectivity, paired t-test was used to analyze the R/S ratio of fluoxetine enantiomers with a null hypothesis of one. The significance level for all statistical analyses used for studies in this thesis was p < 0.05 (two-tailed). Statistical analysis was performed using SPSS software (Microsoft, New York, USA). Unless specifically stated otherwise, all data are reported as average ±	
 standard deviation.  In addition to two-way ANOVA statistical analysis, the relationships between several pharmacokinetic parameters as a function of postnatal age were also investigated using piecewise linear regression analysis with Sigmaplot 11.0 software (Systat Software Inc, California, USA). This analysis used linear regression analysis to calculate the slopes and intercepts for the 2 elements of the curve and the postnatal age at which the breakpoint occur. The equations employed in the analysis were  61  where t, t1, and t2 are the postnatal age in question, and the lowest postnatal age, and the higher postnatal age monitored for the lambs, respectively. T1 is the postnatal age at the breakpoint, and y1, y2, and y3 are the systemic clearance at postnatal ages t1, T1, and t2, respectively.  For the linear regression, the equation of a straight line y=mx + b was used, where m and x were calculated as  62  3. Results	
  	
    3.1 Stereoselective	
  fluoxetine	
  disposition	
  in	
  newborns	
  to	
  1	
  year	
  of	
  age.	
    3.1.1 	
  Developmental	
  plasma	
  fluoxetine	
  pharmacokinetics	
  	
   The average weight (kg) and age (days) of the lambs from newborn (Cohorts1) to ~1 year of age (Cohort 6) at the start of pharmacokinetic experiments are summarized in Table 3.1 and Figure 3.1. Data for individual lambs are presented in Appendix 1.  Table 3.1 Average weight (kg) and age (days) of lambs in cohorts 1 to 6 at the start of pharmacokinetic experiments. Cohort 1 2 3 4 5 6  Age (AVG ± SEM) 5.35 ± 0.292 13.4 ± 0.678 33.2 ± 0.869 98.4 ± 3.14 208 ± 8.91 404 ± 7.76  Weight (AVG±SEM) 4.08 ± 0.415 7.91 ± 0.591 12.77 ± 0.765 22.47 ± 1.56 38.26 ± 2.87 57.2 ± 2.15  Sample Size 12 12 12 12 8 9  63  Figure 3.1 Postnatal weight (kg) plotted as a function of age (days) of the lambs from newborn (Cohort 1) to ~1 year of age (Cohort 6).  The average age of newborn lambs in Cohort #1 was 5.35 ± 1.01 days, and average weight was 4.08 ± 1.44 kg. Pharmacokinetic experiments were performed in a total of 12 lambs with 6 males and 6 females. At 5 minutes after administration of the dose, the average plasma FX concentration was 136.73 ± 17.86 ng/ml and by the end of the experiment at 72 hours the average concentration had declined to 8.46 ± 4.94 ng/ml. The average plasma FX concentration plotted as a function of time in newborns is shown in Figure 3.2. FX concentration had a tri-phasic decline on a logarithmic scale; the data were best fitted by a three-compartmental model based on minimized AIC and SC values. Plasma pharmacokinetic parameters obtained in this cohort are summarized in Table 3.2, and data for each lamb are summarized in Appendix 1. The average clearance of fluoxetine in newborn lambs (Cohort 1) was 6.88 ± 2.20 ml/min/kg with an elimination  64  half-life of 46.10 ± 12.9 hours. The AUC and MRT of fluoxetine were 2624 ± 777 µg*hr/L, and 61.72 ± 14.57 hours, respectively.  Figure 3.2 Newborn (Cohort 1) average plasma fluoxetine concentrations on logarithmic scale as a function of time. Log scale was used for fluoxetine concentrations to best show the tri-phasic decline (AVG ± SEM).  The data suggest that newborn lambs (Cohort 1) have the ability to metabolize fluoxetine to norfluoxetine. Most lambs in Cohort 1 had measurable norfluoxetine (>1ng/mL) in collected plasma within 1 hour after dosing, except for 3 lambs where no norfluoxetine was measurable (< 1ng/mL) in collected plasma samples up to 72 hours after dosing. This may be due to the assay limitations, and norfluoxetine may have been present at levels below 1 ng/ml. The average norfluoxetine AUC in newborns was 469 ± 120 µg*hr/L, with a metabolite to parent ratio (NFX/FX) of 0.179 ± 0.152. In order to estimate NFX AUC, a truncated AUC was used where the area from time zero was calculated to the last time-point, as opposed to estimation to infinity. The rationale was  65  that norfluoxetine concentrations toward the end the collection period did not rapidly decline, and a large portion of AUC would have had to be estimated from the last timepoint to infinity. A truncated estimation of norfluoxetine AUC was also used for cohorts 2 to 6.  Plasma fluoxetine and norfluoxetine concentrations for newborns (Cohort 1) to ~1 year of age (Cohort 6) are shown in Figures 3.3 to 3.8. Comparing the norfluoxetine curves from Cohort 1 to 6, a gradual transformation of the norfluoxetine concentration curve could be observed as the lambs aged and drug disposition abilities developed. In Cohort 1, there were low levels of norfluoxetine produced from the metabolism of fluoxetine with the norfluoxetine concentration rising slowly in plasma and continuing to increase up to the end of the study at 72 hours. In contrast for Cohort 6, plasma norfluoxetine concentrations rose rapidly to a maximum at approximately 10 hours, and declined rapidly thereafter. A tri-phasic decline in fluoxetine plasma concentration was observed on a logarithmic scale for all cohorts; from cohort 1 to 6, a steeper decline during the elimination phase could be observed.  66  Figure 3.3 Cohort 1 average fluoxetine and norfluoxetine plasma concentrations plotted as a function of time (AVG ± SEM).  Figure 3.4 Cohort 2 average fluoxetine and norfluoxetine concentrations plotted as a function of time (AVG ± SEM).  67  Figure 3.5 Cohort 3 average fluoxetine and norfluoxetine plasma concentrations plotted as a function of time (AVG ± SEM).  Figure 3.6 Cohort 4 average fluoxetine and norfluoxeitne plasma concentrations plotted as a function of time (AVG ± SEM).  68  Figure 3.7 Cohort 5 average fluoxetine and norfluoxetine plasma concentrations plotted as a function of time (AVG ± SEM).  Figure 3.8 Cohort 6 average fluoxetine and norfluoxetine plasma concentrations plotted as a function of time (AVG ± SEM).  69  As the lambs grew from newborn (Cohort 1) to ~1 month (Cohort 3) of age, there were no statistically significant differences in the plasma pharmacokinetic data. These data are summarized in Table 3.2.  Table 3.2 Fluoxetine plasma pharmacokinetic data for newborns (Cohorts 1) to ~1 Month of age (Cohort 3) (AVG ± SEM). Pharmacokinetic Parameter FX Sample Size FX AUC (µg*hr/L) FX t1/2(hr) FX Vdss (l/kg) FX Cl (ml/min/kg) FX MRT (hr) NFX AUC (µg*hr/L) NFX/FX Ratio  Cohort #1 (~5 days) 12 2624 ± 224 46.1 ± 3.74 23.3 ± 3.27 6.83 ± 0.65 61.7 ± 4.22 469 ± 96.3 0.179 ± 0.0445  Cohort #2 (~10 days) 12 3394 ± 289 40.2 ± 2.82 29.1 ± 2.53 5.80 ± 1.09 54.8 ± 4.36 1048 ± 186 0.309 ± 0.0744  Cohort #3 (~1 Month) 12 3734 ± 449 35.9 ± 3.12 13.6 ± 1.33 5.20 ± 0.655 47.7 ± 4.53 1090 ± 173 0.292 ± 0.0332  From newborn (Cohort 1) to ~1 month (Cohort 3), the systemic clearance of fluoxetine did not change significantly. AUC increased, though not significantly perhaps due to a greater dose that increased with age (and weight). There were also non-significant trends for decreases in FX t1/2 and MRT values. NFX AUC increased (although not significantly) during this time, which suggests an increasing ability to metabolize fluoxetine to norfluoxetine.  As the lambs grew from ~ 1 month (Cohort 3) to ~3 months (Cohort 4), the plasma pharmacokinetic data indicate a significant maturation in drug disposition ability. These data are summarized in Table 3.3.  70  Table 3.3 Fluoxetine plasma pharmacokinetic data compared between ~1 month (Cohort 3) and ~3 months of age (Cohort 4) (AVG ± SEM). Pharmacokinetic Cohort #3 Parameter (~1 Month) FX Sample Size 12 FX AUC (µg*hr/L) 3734 ± 449 * FX t1/2(hr) 35.9 ± 3.12 * FX Vdss (l/kg) 13.6 ± 1.33 FX Cl (ml/min/kg) 5.20 ± 0.665 * FX MRT (hr) 47.7 ± 4.53 * NFX AUC (µg*hr/L) 1090 ± 173 NFX/FX Ratio 0.292 ± 0.0332 * * denotes a significant age difference  Cohort #4 (~3 Month) 9 1285 ± 173 * 14.5 ± 3.13 * 12.6 ± 1.54 13.8 ± 1.96 * 16.6 ± 3.66 * 1187 ± 289 0.924 ± 0.0941 *  Fluoxetine had a significantly higher clearance and shorter half-life at ~ 3 months compared to ~ 1 month. Correspondingly, a significantly lower AUC and MRT was observed at ~3 months than ~1 month. Notably, the norfluoxetine to fluoxetine ratio had a significant increase at ~3 months (NFX/FX Ratio 0.924 ± 0.282) from ~1 month (0.292 ± 0.115), even though the AUC of norfluoxetine was comparable from ~1 month (NFX AUC 1090 ± 598 µg*hr/L) to ~3 months ((NFX AUC 1187 ± 865µg*hr/L). This dramatic increase in norfluoxetine to fluoxetine ratio during this period was due to the large decrease of FX AUC at ~3 months.  As the lambs grew from ~3 months (Cohort 4) to ~12 months of age (Cohort 6), plasma pharmacokinetic data were comparable between these cohorts and do not suggest significant changes in the disposition of fluoxetine. These data are summarized in Table 3.4.  71  Table 3.4 Fluoxetine and norfluoxetine plasma pharmacokinetic data at ~3 months (Cohort 4) to ~ 12 months of age (Cohort 6) (AVG ± SEM). PK Parameter Sample Size FX AUC (µg*hr/L) FX t1/2 (hr) FX Vdss (L/kg) FX Cl (ml/hr/kg) FX MRT (hr) NFX AUC (µg*hr/L) NFX/FX Ratio  Cohort 4 (~3 Months) 9 1285 ± 173 14.5 ± 3.13 12.6 ± 1.54 13.8 ± 1.96 16.6 ± 3.66 1187 ± 289 0.924 ± 0.0941  Cohort 5 (~6 Months) 8 1650 ± 169 15.5 ± 2.46 10.8 ± 2.51 10.9 ± 1.12 15.6 ± 2.49 1569 ± 229 0.951 ±0.0744  Cohort 6 (~ 12 Months) 10 2983± 551 9.72 ± 1.13 4.10 ± 0.901 7.60 ± 1.33 8.80 ± 1.51 2681 ± 201 0.899 ± 0.126  The systemic clearance of fluoxetine of all lambs is plotted as a function of postnatal age in Figure 3.9. The significant increase from ~1 month to ~3 months can be observed. Further, it is interesting to note the downward trend in systemic clearance from newborn (Cohort 1) to ~ 1 month (Cohort 3), and from ~3 months (Cohort 4) to ~12 months (Cohort 6). The regression line on the graph is estimated using piecewise regression analysis with 2-elements (r2 0.789, p < 0.05); these data are summarized in Table 3.5. A significant breakpoint was observed at 94 ± 16.1 days, which is consistent with statistical analysis using two-way ANOVA.  72  Figure 3.9 Fluoxetine systemic clearance as a function of post-natal age. The regression line was estimated using piecewise regression with 2-elements.  Table 3.5 Fluoxetine systemic clearance as a function of postnatal age using piecewise regression analysis with 2-elements (AVG ± SEM). Region I  Region 2 BP (d) Slope Intercept Slope Intercept 94 ± 16.1 0.105 ± 0.0432* 4.27 ± 1.23 -0.0089 ± 0.0142 13.4 ± 2.12 *denotes slope was significantly different than 0.  Vdss for fluoxetine was plotted as a function of postnatal age for each lamb in Figure 3.10. There is a downward trend in Vdss as the lambs aged. The line was estimated using linear regression (r2 = - 0.474, p = 0.745). The negative correlation coefficient suggests a negative correlation, or decrease in Vdss, as the lambs aged.  73  Figure 3.10 Fluoxetine volume of distribution at steady state as a function of post-natal age. The line was estimated linear regression (r 2= -0.474, p =< ).001).  The plasma metabolite to parent compound ratio (NFX/NFX) for each lamb was plotted as a function of postnatal age and analyzed using piecewise regression analysis (R2 = 0.765, p < 0.025); this is summarized in Figure 3.11 and Table 3.6. A significant breakpoint was observed at 110 ± 17.7 days. This value is not significantly different from the breakpoint value of 94.6±16.1 for FX clearance (see Table 3.5).  74  Figure 3.11 Plasma NFX/FX AUC ratio as a function of postnatal age. The regression line was estimated using piecewise linear regression with 2-elements. Table 3.6 Piecewise regression analysis with 2-elements of plasma NFX/FX AUC ratio as a function of postnatal age (AVG ± SEM). Region I BP (d) Slope Intercept 110 ± 17.7 0.0107 ± 0.0063* 0.106 ± 0.092 *denotes slope is significant different from 0.  Region 2 Slope Intercept 6E5 ± 0.0193 1.04 ± 0.96  Plasma metabolite to parent compound ratio (NFX/FX) for each lamb was plotted as a function of clearance in Figure 3.12. As clearance increases, there is a proportional increase in NFX/FX ratio. The line was estimated using linear regression (r2 = 0.786, p < 0.001).  75  Figure 3.12 Plasma metabolite to parent compound ratio (NFX/FX) clearance. The line was estimated using linear regression (r 2= 0.786, p < 0.001).  3.1.2 Stereoselective	
  plasma	
  pharmacokinetics	
   The stereoselective plasma pharmacokinetic data for all lambs are presented in Appendix 1. To estimate stereoselective disposition, the S/R ratios of the plasma pharmacokinetic data were calculated. The S/R ratios of plasma pharmacokinetic data for newborns are presented in Figure 3.13.  76  Figure 3.13 Newborn (Cohort 1) S/R ratio of plasma pharmacokinetic data (AVG ± SEM); the asterisk (*) denotes significant difference from 1.  The plasma pharmacokinetics of fluoxetine in the newborns (Cohort 1) is stereoselective. Plasma concentrations of fluoxetine enantiomers are plotted as a function of time in Figure 3.14. SFX concentrations were significantly greater than RFX in plasma. Using the Students t-test and a null hypothesis of 1, the S/R ratios of plasma pharmacokinetic parameters were significantly different than 1. The clearance of SFX (0.57 ± 0.18 L/hr/kg) was significantly lower than RFX (1.52 ± 0.43 L/hr/kg). Correspondingly, AUC and MRT were greater than RFX and the half-life of SFX was longer. These data indicate that the disposition of racemic fluoxetine is enantioselective, and that the elimination of RFX is more rapid than SFX in these newborns.  77  Figure 3.14 Average plasma fluoxetine enantiomer concentrations as a function of time in newborns (AVG ± SEM).  Plasma norfluoxetine concentrations were not significantly different between enantiomers in newborns (Cohort 1). While the AUC of SNFX (SNFX 227.8 ± 14.7) was greater than the AUC for RNFX (186.32 ± 10.9), this difference was not significant. The norfluoxetine to fluoxetine ratio is statistically significant between enantiomers – SNFX/FX was 0.116 ± 0.082, and RNFX/FX was 0.261 ± 0.184 perhaps due to the fact that SFX AUC was significantly greater than RFX AUC. Further, as mentioned previously norfluoxetine AUCs may be underestimated due to the fact that its concentration in plasma was still rising at the last time point.  As the newborns aged to ~12 months of age, plasma fluoxetine pharmacokinetics continued to be stereoselective. The S/R ratio of plasma pharmacokinetic data from ~ 10 days (Cohorts 2) to ~ 12 months (Cohort 6) are summarized in Figures 3.15 to 3.19. Data for individual lambs are presented in Appendix 1. The S/R ratios of plasma  78  pharmacokinetic parameters were significantly different than 1 suggesting stereoselective disposition of fluoxetine, with the disposition of R more rapid than S throughout the first year of life.  Figure 3.15 Cohort 2 S/R ratio of plasma pharmacokinetic data (AVG ± SEM); the asterisk (*) denotes significant difference from 1.  79  Figure 3.16 Cohort 3 S/R ratio of plasma pharmacokinetic data (AVG ± SEM); the asterisk (*) denotes significant difference from 1.  Figure 3.17 Cohort 4 S/R ratio of plasma pharmacokinetic data (AVG ± SEM); the asterisk (*) denotes significant difference from 1.  80  Figure 3.18 Cohort 5 S/R ratio of plasma pharmacokinetic data (AVG ± SEM), the asterisk (*) denotes significant difference from 1.  Figure 3.19 Cohort 6 S/R ratio of plasma pharmacokinetic data (AVG ± SEM), the asterisk (*) denotes significant difference from 1.  81  The S/R ratios of plasma pharmacokinetic parameters were not significantly different between cohorts; this indicates that in terms of stereoselective disposition there were no changes throughout the first year of life. Figure 3.20 illustrates the S/R ratios of fluoxetine systemic clearance as a function of postnatal age of all lambs. The line was estimated using linear regression (r 2= -0.032, p = 0.496) and suggests a poor association, or lack of change, of FX Cl S/R ratio as the lambs aged.  Figure 3.20 Systemic clearance S/R ratio as a function of postnatal age. The regression line was estimated using linear regression (r2 = -0.032, p 0.496).  3.1.3 Developmental	
  urinary	
  pharmacokinetics	
   Urine pharmacokinetic data for individual lambs are presented in Appendix 2. The weight adjusted renal clearance of fluoxetine as the lambs aged from newborn (Cohort 1) to ~1 year of age (Cohort 6) are illustrated in Figure 3.21. A significant  82  increase in fluoxetine renal clearance was observed as the lambs aged from ~1 months (Cohort 3) to ~3 months (Cohort 4).  Figure 3.21 Fluoxetine weight adjusted renal clearance from newborn (Cohort) 1 to ~1 year of age (Cohort 6). Letters a and b denotes significant differences (AVG ± SEM).  A representative graph of urine fluoxetine and TFMP levels recovered in newborns (Cohort 1) is illustrated in Figure 3.22. The accumulated amount of the fluoxetine recovered in newborn (Cohort 1) urine samples was 132.7 ± 8.25 µg, or 2.86 ± 0.28% of the dose. In addition to unchanged fluoxetine, its metabolite TFMP was also observed in the urine of newborns; 41.2 ± 4.30 µg of TFMP was recovered, or 1.82 ± 0.46% the dose. No other metabolites were observed in the collected samples. In total, 4.68 ± 0.863% of FX and its metabolite TFMP was recovered from the administered dose in newborns. FX and TFMP levels did not appear to reach a plateau by 72 hours, which may lead to underestimation of their renal PK parameters.  83  Figure 3.22 Representative urine fluoxetine and TFMP levels as a function of time in newborn lambs (AVG ± SEM).  As the newborns aged to ~10 days (Cohort 2), only FX and its TFMP metabolite were observed in urine. The amount of FX and TFMP recovered at ~ 10 days (Cohort 2) was greater than newborns, though not significantly. The amount of FX recovered in urine was 147.7 ± 4.15 µg, or 2.03 ± 0.225 % of dose, and the amount of TFMP recovered was less than its parent compound at 83.1 ± 2.90 µg, or 1.20 ± 0.51% of dose. Overall, the percent of dose recovered from FX and its TFMP metabolite was 3.23 ± 0.873%.  Interesting observations in the urine samples were observed as the lambs aged to ~ 1 month (Cohort 3) as additional fluoxetine Phase I and II metabolites were observed in urine. Overall, the percent of dose recovered for FX and all of its metabolites was 2.94 ± 0.942%. The amount of FX and TFMP recovered in urine were greater than Cohort 2, though not significantly. The amount of FX recovered in urine was 155.98 ± 8.065 µg, or  84  1.29 ± 0.155 % of dose. The amount of TFMP recovered in urine was less at 108.5 ± 9.69 µg, or 0.88 ± 0.16 % of dose.  Norfluoxetine was observed in the urine at ~1 month (Cohort 3). The amount recovered was 34.4 ± 5.52 µg, or 0.269 ± 0.0465 % of the administered fluoxetine dose. A plot of FX and Phase I metabolites NFX and TFMP concentrations in urine as a function of time is shown in Figure 3.23. FX, NFX, and TFMP levels did not appear to reach a plateau by 72 hours, which may lead to underestimation of their renal PK parameters. The level of the parent compound fluoxetine was highest in urine, followed by TFMP and norfluoxetine at ~ 1 month (Cohort 3) of age.  Figure 3.23 Fluoxetine and Phase I metabolites norfluoxetine and TFMP concentrations in urine samples as a function of time at ~ 1 month of age (Cohort 3) (AVG ± SEM).  Phase II metabolites were observed in urine at ~ 1 month (Cohort 3). The amount of FX-glucuronide recovered was 30 ± 3.205 µg, or 0.195 ± 0.023 % of dose while the 85  amount of NFX-glucuronide recovered was 58.1 ± 9.3 µg, or 0.31 ± 0.033 % of dose. A representative plot of FX and NFX glucuronides is shown in Figure 3.24. FX and NFX glucuronide levels did not appear to reach a plateau by 72 hours, which may lead to underestimation of their renal PK parameters. The level of NFX-glucuronide was higher than FX-glucuronide in urine at ~1 month (Cohort 3).  Figure 3.24 Fluoxetine and norfluoxetine glucuronide concentrations in urine as a function of time at ~1 month of age (Cohort 3) (AVG ± SEM).  At ~1 month (Cohort 3), the level of unchanged FX in urine was higher than its metabolites. From Phase I metabolism, the level of TFMP was higher than NFX. Metabolite levels from Phase I metabolism were quantitatively higher than Phase II. From Phase II metabolism, the level of NFX-glucuronide was higher than FXglucuronide. `  86  As the lambs aged to ~3 months (Cohort 4), a significant increase in the levels of fluoxetine and its metabolites were observed in urine, with the exception of TFMP where an increase was observed, though not significant. In total, the amount of FX and metabolites recovered in urine was 216.5 ± 11.0 µg, or 1.03 ± 0.111 % of dose. A comparison of fluoxetine and its metabolite levels in urine between ~ 1 month (Cohort 3) and ~ 3 months (Cohort 4) is shown in Figure 3.25 (see also Fig. 3.26).  * *  *  *  *  *  *  *  Figure 3.25 Comparison of cummulative urine levels of fluoxetine and its metabolites between ~1 month (Cohort 3) an ~ 3 months (Cohort 4) (AVG ± SEM). The asterisk (*) denotes significant differences.  In contrast to the ~ 1 month (Cohort 3) old lambs, as the lambs matured to ~ 3 months (Cohort 4) of age the level of norfluoxetine exceeded that of FX. Similarly for Phase II metabolism the levels of FX- and NFX-glucuronide exceeded that of FX. Figure 3.26 compares the amount of fluoxetine and its metabolites recovered in urine from ~ 1 month (Cohort 3) to ~12 months (Cohort 6). As the lambs grew to ~ 6 months, and ~ 12  87  months, an increase in fluoxetine and its metabolites in urine was observed, however this change was not significant.  *  *  *  * * *  *  *  Figure 3.26 Amount recovered in urine for fluoxetine, norfluoxetine, fluoxetineglucuronide, and norfluoxetine-gluruconide compared from ~1 month (Cohort 3) to ~12 months (Cohort 6). The asterisk (*) denotes significant difference (AVG ± SEM). Considering the Phase I pathways as the lambs aged to ~1 year of age, norfluoxetine levels continued to be greater than TFMP and the parent compound. In contrast, from Phase II metabolism norfluoxetine-glucuronide levels were comparable to fluoxetine-glucuronide, but greater than the parent compound.  3.1.4 Stereoselective	
  urinary	
  pharmacokinetics	
  	
   Renal elimination of FX was stereoselective in the newborn. The urine concentration of SFX was consistently greater than RFX. A representative plot of the 88  urine concentrations of FX enantiomers for newborns as a function of time is shown in Figure 3.27. The S/R ratios for the fluoxetine urine pharmacokinetics parameters were significantly different than 1. The S/R ratio for the amount of FX recovered was 1.63 ± 0.42, and renal clearance the ratio was 1.28 ± 0.062.  Figure 3.27 Representative urine concentrations of fluoxetine enantiomers as a function of time in newborns (AVG ± SEM).  As mentioned previously, at 1 month (Cohort 3), norfluoxetine and glucuronide conjugates of fluoxetine and norfluoxetine were observed in urine. The renal elimination of norfluoxetine was stereoselective, and a comparison of enantiomer levels is presented in the following figures. Figure 3.28 illustrates the representative urine concentrations of fluoxetine and norfluoxetine enantiomers as a function of time; the levels of SFX are consistently greater than RFX in urine. Similarly, the levels of SNFX are consistently greater than RNFX in urine.  89  Figure 3.28 Representative urine fluoxetine and norfluoxetine enantiomers, and TFMP concentrations as a function of time. at ~ 1 month (Cohort 3) (AVG ± SEM).  Figure 3.29 illustrates the representative urine concentrations of fluoxetine and norfluoxetine enantiomer glucuronides as a function of time. In contrast to FX and NFX, the enantiomer levels of FX-glucuronide and NFX-glucuronide are comparable in urine. The renal elimination of FX- and NFX-glucuronides is not stereoselective.  90  Figure 3.29 Representative urine fluoxetine and norfluoxetine enantiomer glucuronide concentrations as a function of time at ~ 1 month (Cohort 3) (AVG ± SEM).  Developmentally there did not appear to be significant changes in the stereoselective disposition of FX as the lambs aged from Cohort 1 to 6. Figure 3.30 summarizes the S/R ratios for urine pharmacokinetic data where S/R ratios are comparable as the lambs aged from newborn (Cohort 1) to ~1 year of age (Cohort 6).  91  S/R Ratio Figure 3.30 S/R ratio of urine pharmacokinetic parameters compared between cohorts 1 to 6 (AVG ± SEM). 3.1.5 Developmental	
  and	
  stereoselective	
  fluoxetine	
  and	
  norfluoxetine	
  plasma	
   protein	
  binding	
   Fluoxetine binding to plasma protein measured by equilibrium dialysis for Cohorts 1 to 6 is summarized in Figure 3.31.  92  Figure 3.31 Unbound fraction of fluoxetine in plasma for cohorts 1 to 6; Columns with different letters above them are significantly different (AVG ± SEM).  As the lambs grew from newborn (Cohort 1) to ~1 year of age (Cohort 6), the binding of fluoxetine to plasma proteins increased. The fraction unbound of Cohorts 1 and 2 are significantly greater than cohorts 4 and 6.  To investigate the stereoselective binding of FX to plasma protein, the S/R ratio of S- and R-FX fraction unbound to plasma protein was calculated. These data are summarized in Figure 3.32. Overall, the S/R ratio of the fraction unbound was significantly less than a null hypothesis of 1 for all cohorts. The fraction unbound of SFX was in general less than RFX; this was consistent with plasma and urine data where a greater level of S was observed. There are no significant differences in the S/R ratio of FX fraction unbound between cohorts.  93  Figure 3.32 S/R ratio of unbound fraction of fluoxetine in plasma for cohorts 1 to 6 (AVG ± SEM). The asterisk (*) denotes statistical significance from null hypothesis of 1.  Stereoselective systemic clearance values were presented in section 3.1.2. Systemic clearance represents the clearance of both bound and unbound drug. However, only the unbound drug fraction is free to undergo metabolism and elimination. By knowing the fraction of drug unbound, it is possible to determine free clearance, or clearance for the unbound fraction of drug. Free clearances of S- and R-FX were calculated and are summarized in Table 3.7. Using the Paired Students t-test and a null hypothesis of 0 (no differences between S- and R-FX free clearance), the average difference between S- and R-FX free clearance of all Cohorts was 0.01 ±	
 0.043, and a t value of 0.278 was obtained (p > 0.05); these data suggest that after factoring in the stereoselective binding of FX to plasma proteins, there are no significant differences between the calculated free clearances of S- and RFX.  94  Table 3.7 Stereoselective free clearance for cohorts 1 to 6. Cohort 1 2 3 4 5 6  RFX (ml/min/kg) 197.46 198.26 228.14 1131.30 911.60 432.13  SFX (ml/min/kg) 180.41 176.05 211.39 1191.04 827.08 528.35  S/R 0.914 0.887 0.926 1.25 0.907 1.22  	
   Norfluoxetine plasma protein binding was also determined by equilibrium dialysis. These data are summarized in Figure 3.33. Similar to fluoxetine, the binding of norfluoxetine to plasma protein appeared to increase as the lambs aged from newborn to ~1 year of life. The unbound fraction of fluoxetine in plasma is significantly greater for cohorts 1 and 2 than cohorts 4 to 6.  Figure 3.33 Norfluoxetine fraction unbound in plasma for cohorts 1 to 6Columns with different letters above them are significantly different based on two-way ANOVA (age, sex) (AVG ± SEM).  95  The binding of norfluoxetine to plasma proteins is also stereoselective for each cohort. The S/R ratio of the unbound fraction of norfluoxetine in plasma is summarized in Figure 3.34. Similar to the fluoxetine results, the binding of SNFX was greater than RNFX. Using the Students t-test and a null hypothesis of 1, the S/R ratio was significantly less than 1 (t = 3.693, p < 0.025). This corroborates the plasma and urine pharmacokinetics data where levels of SNFX were greater than RNFX.  Figure 3.34 S/R ratio of norfluoxetine unbound fraction for Cohorts 1 to 6. (AVG ± SEM). The asterisk (*) denotes statistical significance from null hypothesis of 1.  3.1.6 Developmental	
  plasma	
  protein	
  levels	
   The AAG plasma concentration data for cohorts 1 to 6 is summarized in Figure 3.35. There appeared to be an increase in the level of AAG as the lambs grew from newborn (Cohort 1) to ~1 year of age (Cohort 6). AAG concentrations for cohorts 1 and 2 are significantly lower than 4 to 6.  96  Figure 3.35 AAG plasma concentrations for cohorts 1 to 6; Columns with different letters above them are significantly different based on two-way ANOVA (age, sex) (AVG ± SEM).  Total plasma protein concentration for cohorts 1 to 6 are summarized in Figure 3.36. Similar to AAG, as the lambs aged from newborn (Cohort 1) to ~1 year of age (Cohort 6), total plasma protein level increased total plasma protein concentrations for cohorts 1 and 2 are significantly less than cohorts 4 to 6.  97  Figure 3.36 Total plasma protein concentrations for cohorts 1 to 6; Columns with different letters above them are significantly different based on two-way ANOVA (age, ) (AVG ± SEM).  98  3.1.7 Sex	
  differences	
  in	
  fluoxetine	
  disposition	
  from	
  newborn	
  to	
  1	
  year	
  of	
  age.	
   The average weight (kg) and age (days) of male and female lambs from newborn (Cohorts1) to ~ 1 year of age (Cohort 6) at the start of pharmacokinetics experiments are summarized in Table 3.8 and Figure 3.37. Data for individual lambs are presented in Appendix 1. There were no significant sex differences in the weight and age of male and female lambs within each cohort at the start of pharmacokinetic experiments.  Table 3.8 Average weight (kg) and age (days) of lambs in cohorts 1 to 6 at the start of pharmacokinetic experiments (AVG ± SEM). Age  Male Female Male + Female Male Female Male + Female Male Female Male + Female Male Female Male + Female Male Female Male + Female Male Female Male + Female  Weight Cohort #1 5.38 ± 0.375 4.01 ± 0.772 5.32 ± 0.478 4.17 ± 0.410 5.35 ± 0.412 4.08 ± 0.588 Cohort #2 12.17 ± 0.306 7.62 ± 0.221 14.83 ± 1.05 8.22 ± 1.85 13.42 ± 0.959 7.91 ± 0.837 Cohort #3 31.42 ± 0.465 12.84 ± 0.249 35.00 ± 1.36 13.38 ± 1.53 33.20 ± 1.23 12.77 ± 1.08 Cohort #4 95.33 ± 0.514 22.97 ± 1.71 100.00 ± 3.91 25.48 ± 3.25 98.43 ± 3.14 22.47 ± 1.56 Cohort #5 227.53 ± 6.31 39.55 ± 3.29 190.01 ± 9.83 36.98 ± 5.17 208.75 ± 8.91 38.26 ± 2.87 Cohort #6 399 ± 12.1 60.6 ± 1.91 405 ± 12.1 52.0 ± 2.89 404 ± 7.65 57.2 ± 2.44  Sample Size 6 6 12 6 6 12 6 6 12 6 6 12 4 4 8 5 4 9 99  Figure 3.37 Postnatal weight (kg) as a function of age (days) for male and female lambs.  When plasma pharmacokinetic data for newborn lambs (Cohort 1) were compared between sexes, a compelling difference in fluoxetine pharmacokinetics was observed based on sex averages and using two-way AVOVA with age and sex as dependent variables. Data for cohorts 1 to 6 are summarized in Table 3.9, and for individual lambs in Appendix 1. Average plasma fluoxetine concentrations as a function of time for male and female newborn lambs are shown in Figure 3.38.  100  Table 3.9 Male and female newborn (Cohort 1) fluoxetine plasma pharmacokinetic data (AVG ± SEM). Pharmacokinetic Parameter Male FX Sample Size 6 FX AUC (µg*hr/L) 3313.9 ± 162 * FX t1/2(hr) 59.4 ± 2.11 * FX Vdss (l/kg) 23.2 ± 6.54 FX Cl (ml/hr/kg) 4.83 ± 0.231 * FX MRT (hr) 78.9 ± 6.28 * NFX AUC 216.9 ± 13.5 * NFX/FX Ratio 0.07 ± 0.0735 * * denotes statistical difference between sex.  Female 6 1934.6 ± 71.7 * 32.7 ± 1.35 * 23.4 ± 1.84 8.67 ± 0.361 * 44.5 ± 1.77 * 439.9 ± 28.9 * 0.23 ± 0.0531 *  Figure 3.38 Average plasma fluoxetine concentrations as a function of time for male and female newborn lambs (AVG ± SEM) (sample size: male = 6; female = 6).  From right after administration of the dose, plasma fluoxetine concentrations in the female appeared to be lower than those in the male, and the values were significantly different between sex from 30 minutes onwards until 36 hours. Fluoxetine concentrations declined more rapidly for females than males. Fluoxetine clearance was significantly  101  higher in females than males, and elimination half-life was significantly shorter in females than males; females had greater metabolic capacity for fluoxetine than males. Correspondingly, fluoxetine had a lower AUC and MRT in females, suggesting they had a lower exposure to this drug than males at this age. Neither the average age nor weight of females and males was significantly different at the start of their experiments; these data are summarized in Table 3.8.  Plasma fluoxetine pharmacokinetics data for males and females in cohorts 1 to 6 are summarized in Table 3.10. Statistically significant differences between sex are denoted by an asterisk (*). No statistically significant sex differences in plasma pharmacokinetics were observed beyond Cohort 1.  Table 3.10 Plasma pharmacokinetics data for males and females in cohorts 1 to 6 (AVG ± SEM). Cohort 1 FX AUC (µg*hr/L) FX Elimination Half-life (hr) FX Volume of Distribution (L/kg) FX Clearance (ml/hr/kg) FX Mean Residence Time (hr) NFX/FX AUC Ratio Cohort 2 FX AUC (µg*hr/L) FX Elimination Half-life (hr) FX Volume of Distribution (L/kg) FX Clearance (ml/hr/kg) FX Mean Residence Time (hr) NFX/FX AUC  Female (n=6) 1934.6 ± 71.7 * 32.7 ± 1.35 * 23.4 ± 1.84 8.67 ± 0.361 * 44.5 ± 1.77 * 439.9 ± 28.9 * Female (n=6) 2910.3 ± 417 46.09 ± 4.83 46.13 ± 1.05 7.00 ± 2.09 58.34 ± 7.57 0.349 ± 0.142  Male (n=6) 3313.9 ± 162 * 59.4 ± 2.11 * 23.2 ± 6.54 4.83 ± 0.231 * 78.9 ± 6.28 * 216.9 ± 13.5 * Male (n=6) 3879.2 ± 316 54.38 ± 2.26 42.02 ± 2.71 4.50 ± 0.340 55.17 ± 3.17 0.252 ± 0.0531 102  Cohort 3 Female (n=6) FX AUC (µg*hr/L) 3052.3 ± 332 FX Elimination Half-life (hr) 29.41 ± 3.35 FX Volume of Distribution (L/kg) 12.35 ± 1.04 FX Clearance (ml/hr/kg) 5.67 ± 0.612 FX Mean Residence Time (hr) 38.35 ± 5.69 NFX/FX AUC 0.293 ± 0.036 Cohort 4 Female (n=6) FX AUC (µg*hr/L) 1479.4 ± 135 FX Elimination Half-life (hr) 18.1 ± 3.29 FX Volume of Distribution (L/kg) 13.8 ± 2.07 FX Clearance (ml/hr/kg) 11.7 ± 0.951 FX Mean Residence Time (hr) 19.68 ± 1.976 NFX/FX AUC 0.941 ± 0.147 Cohort 5 Female (n=4) FX AUC (µg*hr/L) 1448.9 ± 163 FX Elimination Half-life (hr) 15.02 ± 3.56 FX Volume of Distribution (L/kg) 12.43 ± 3.52 FX Clearance (ml/hr/kg) 11.8 ± 1.17 FX Mean Residence Time (hr) 17.34 ± 4.14 NFX/FX AUC 1.08 ±0.0715 Cohort 6 Female (n=4) FX AUC (µg*hr/L) 1671.5 ± 241 FX Elimination Half-life (hr) 8.63 ± 0.991 FX Volume of Distribution (L/kg) 5.03 ± 0.682 FX Clearance (ml/hr/kg) 10.3 ± 1.42 FX Mean Residence Time (hr) 8.06 ± 0.851 NFX/FX AUC 0.987 ± 0.252 * denotes statistical difference between sex  Male (n=6) 4416.2 ± 777 42.44 ± 3.80 14.76 ± 2.53 4.50 ± 0.886 57.07 ± 4.88 0.289 ± 0.0600 Male (n=6) 1601.9 ± 326 21.48 ± 5.26 16.13 ± 1.46 12.2 ± 2.18 26.32 ± 6.70 0.907 ± 0.0849 Male (n=4) 1851.8 ± 281 14.91 ± 3.66 9.33 ± 3.94 9.83 ± 2.31 14.99 ± 3.17 0.818 ±0.0935 Male (n=6) 2435.5 ± 671 8.36 ± 0.735 5.08 ± 1.49 9.17 ± 1.91 8.36 ± 1.56 0.842 ± 0.143  In addition to two-way ANOVA statistical analysis, sex differences were evaluated using various regression analyses, such as logarithmic and multi-element piecewise regression. Piecewise linear regression analysis with 2-elements best fit the data (r2 = 0.722, p < 0.05). This analysis is summarized in Figure 3.39 and Table 3.11.  103  Figure 3.39 Piecewise regression analysis of fluoxetine clearance as a function of postnatal age.  Table 3.11 Break point, slopes, and intercept values using piecewise 2-element regression analysis of the relationship between fluoxetine systemic clearance and postnatal age for male and female lambs (AVG ± SEM). BP (d)  Region I  Slope Intercept Male 94.0 ± 26.1 0.0492 ± 0.0022* 3.38 ± 1.7* Female 110.0 ± 31.8 0.325 ± 0.059* 6.05 ± 1.9* * denotes significant difference between sex  Region 2 Slope Intercept -0.0122 ± 0.0036* 14.1 ± 2.8 -.00737 ± 0.00031* 13.8 ± 3.4  The breakpoints occurred at 94.0 ± 26.1 days for males, and 110.0 ± 31.8 days for females. These breakpoints are not significantly different between sex. However, it is interesting to note that the slopes for males are significantly steeper than females. These slope differences suggest developmental changes in clearance for males were more rapid than females as the lambs aged. In addition, both before and after the breakpoint, the clearance values in females tended to be higher than those in the males. This is also  104  indicated in Table 3.20; for every cohort, except cohort 4 (age closest to the breakpoint), the mean FX clearance value in females is higher than the value in males, Comparison of the female and male mean clearance values using the paired t test demonstrated that as a group females have a higher clearance value (t = 2.8137, p < 0.025).	
    Stereoselective plasma pharmacokinetics data for male and females lambs from newborn (Cohort 1) to ~1 year of age (Cohort 6) are summarized in Table 3.19. Plasma pharmacokinetic data are stereoselective and comparable between sex at each cohort. Based on two-way ANOVA analysis with age and sex as the dependent variables, stereoselectivity is comparable between sex as the lambs aged from newborn to ~1 year of age.  3.1.8 Stereoselective	
  urinary	
  data	
  compared	
  between	
  sex	
   Stereoselective urinary data for individual male and females from newborn (Cohort 1) to ~1 year of age (Cohort 6) are summarized in Appendix 2. In the newborn, the amount of FX recovered in urine was significantly greater for females (151.8 ± 15.95 µg) than males (113.9 ± 10.6 µg) in Cohort 1. The weight adjusted renal clearance (CLr) of FX for females (0.265 ± 0.0623 ml/min/kg ) was significantly higher than males (0.126 ± 0.0408 ml/min/kg). A representative plot of newborn male and female FX concentration in urine samples as a function of time is shown in Figure 3.40.  105  Figure 3.40 Representative fluoxetine concentrations in urine samples of male and female newborn lambs.  The renal excretion of fluoxetine compared between sex is illustrated in Figure 3.41. Female newborns excreted more FX than males. As the lambs aged to ~1 year of age, renal excretion of fluoxetine was comparable between sex.  Figure 3.41 Renal excretion of FX compared between males and females in cohorts 1 to 6 (AVG ± SEM). The asterisk (*) denotes significant sex differences.  106  The CLr of FX compared between sex for cohorts 1 to 6 is illustrated in Figure 3.42. Beyond newborns (Cohorts 1), no further sex differences were observed as the lambs aged to ~1 year of age.  Figure 3.42 Fluoxetine weight-adjusted renal clearance compared between males and females in cohorts 1 to 6 (AVG ± SEM). The asterisk (*) denotes significant sex differences. The renal clearance of fluoxetine plotted as a function of systemic clearance for individual lambs is shown in Figure 3.43. These data suggest that the contribution of renal clearance to total systemic clearance does not change with age and is not different between sexes. However, it is different between R and S isomers.  107  Figure 3.43 Fluoxetine renal clearance plotted as a function of systemic clearance for all lambs. In contrast to FX in the newborn, the amount of TFMP recovered in urine samples was comparable between females (79.2 ± 7.80 µg) and males (72.5 ± 9.31 µg). Correspondingly, the %dose of TFMP recovered in the urine sample was comparable between sex: for females this was 2.10 ± 0.404%, and 1.50 ± 0.492% for males. Because plasma TFMP PK parameters were not estimated, there was insufficient information to estimate the renal clearance for TFMP. A representative plot of male and female TFMP urine accumulation is shown in Figure 3.44.  108  Figure 3.44 Representative TFMP concentrations in urine samples for male and female newborn lambs. Renal excretion of TFMP compared between males and females as the lambs aged from newborn (Cohort 1) to ~1 year of age (Cohort 6) is illustrated in Figure 3.45. No sex differences in TFMP renal excretion could be observed.  Figure 3.45 TFMP urinary accumulation compared between males and females in cohorts 1 to 6 (AVG ± SEM).  109  As mentioned previously, additional Phase I (NFX) and Phase II (FX and NFX glucuronides) were observed as the lambs aged to ~ 1 month (Cohort 3). Male and female renal excretion of NFX is illustrated in Figure 3.47. The renal excretion of NFX was comparable between sex.  Figure 3.46 NFX urinary accumulation in Cohorts 3 to 6 compared between sex (AVG ± SEM). The renal excretions of FX- and NFX-glucuronide are shown in Figures 3.47 and 3.48. Student’s Paired t-test. For Cohort 3, the renal excretion of FX-glucuronide was significantly different between sex (unpaired t test, t = 3.89, p = 0.003). Similarly, the renal excretion of NFX-glucuronide was significantly different between sex (t = 6.685, p = 0.001). This difference was not present in the other cohorts.  110  Figure 3.47 FX-glucuronide urinary accumulation in Cohorts 3 to 6 compared between sex (AVG ± SEM).  Figure 3.48 NFX-glucuronide urinary accumulation in Cohorts 3 to 6 compared between sex. (AVG ± SEM).  To evaluate potential sex differences in the stereoselective renal clearance of fluoxetine, the S/R ratio of FX Clr was evaluated between sex. Data for individual lambs are presented in Appendix 2. The S/R ratio of FX Clr compared between sex for Cohorts  111  1 to 6 are illustrated in Figure 3.49. Data are comparable between sex and do not suggest sex differences in the stereoselective renal elimination of fluoxetine.  Figure 3.49 S/R ratio of FX weight-adjusted renal clearance compared between sex for cohorts 1 to 6 (AVG ± SEM) . Similarly, the S/R ratio of the urinary accumulation of fluoxetine was comparable between sex. These data are illustrated in Figure 3.50.  112  Figure 3.50 S/R ratio of the amount of fluoxetine recovered in urine compared between sex for cohorts 1 to 6 (AVG ± SEM). The stereoselective renal elimination of norfluoxetine was not significantly different between sex. These data are illustrated in Figure 3.51.  Figure 3.51 S/R ratio of NFX urinary accumulation in urine for cohorts 1 to 6 (AVG ± SEM).  113  3.1.9 Fluoxetine	
  and	
  norfluoxetine	
  plasma	
  protein	
  binding,	
  blood	
  pH	
  and	
   temperature	
  compared	
  between	
  sex	
   Fluoxetine binding to plasma protein for males and females in Cohorts 1 to 6 are shown in Figure 3.51. In the newborns (Cohort 1), the fraction of FX unbound to plasma proteins was 13.1 ± 1.66% in females, and 10.3 ± 1.40% in males; though FX was bound to plasma proteins to a greater extent in males, this difference was not significantly different. Moreover, no sex differences in the binding of FX to plasma proteins were observed in older cohorts.  Figure 3.52 Unbound fraction of fluoxetine in plasma for male and female lambs from cohort 1 to 6 (AVG ± SEM).  In section 3.2.1, fluoxetine systemic clearance was presented for male and female lambs. Systemic clearance represents the clearance of both bound and unbound drug. However, only the unbound drug fraction is free to undergo metabolism and elimination.  114  By knowing the fraction of drug unbound, it is possible to determine free clearance, or clearance for the unbound fraction of drug. Calculated free clearance values for males and females in Cohorts 1 to 6 are presented in Table 3.12. The free clearance of fluoxetine is consistently greater in females compared to males in Cohorts 1 to 6. Using the Student’s paired t-test, this female and male difference in free clearance is statistically significant (t=3.697; p<0.025). Table 3.12 Fluoxetine free clearance values for males and females in cohorts 1 to 6. Cohort 1 2 3 4 5 6  Male (ml/min/kg) 40.30 46.94 148.04 520.30 313.84 309.52  Female (ml/min/kg) 86.13 63.48 184.88 581.56 331.57 407.01  Norfluoxetine plasma protein binding for males and females are shown in Figure 3.52. The fraction unbound of NFX in females was higher than males for Cohorts 1 to 6. Using the Student’s Paired t-test to compare the unbound fraction at each cohort between sex, a significant difference was observed (t = 3.07, p = 0.025).  115  Figure 3.53 Unbound fraction of norfluoxetine compared between age and sex (AVG ± SEM).  3.1.10 	
  Plasma	
  protein	
  levels,	
  temperature	
  and	
  pH	
  compared	
  between	
  sex	
   AAG plasma concentration for cohorts 1 to 6 for males and females are summarized in Figures 3.53. There do not appear to be significant sex differences in AAG plasma concentrations.  116  Figure 3.54 Plasma AAG concentration compared by age and sex (AVG ± SEM).  Total plasma protein concentration for cohorts 1 to 6 for males and females are summarized in Figures 3.54. There do not appear to be significant sex differences in total protein levels for any of the cohorts.  Figure 3.54 Total plasma protein level compared by age and sex (AVG ± SEM).  117  The dissertation work of Tuan Anh-Nguyen, a PhD student in our group, explored the physiological outcomes of postnatal exposure to fluoxetine. As part of her work, blood pH and body temperature were measured at 15 minutes prior to dosing, and at 5, 15, 30, 60, and 120 minutes after dosing. In order to observe drug effects, blood pH and temperature were measured with an injection of sterile saline for control experiments, and with FX for FX experiments. The influence of blood pH and temperature on plasma protein binding has been cited in the literature(Siggaard-Andersen 1971; Velez, Myers, and Guber 1985; Harrisons 2008); thus, these data were analyzed for potential effects on plasma protein binding, and thus pharmacokinetics in the current studies; these detailed data are presented in Appendix 3. (Tuan-Anh Nguyen unpublished data)  No significant differences were observed between control and FX experiments for blood pH and temperature. No age related differences were observed between cohorts 1 to 6. However, a significant difference was observed between sex for blood pH in the newborns (Cohort 1) for blood samples collected up to 15 minutes after dosing; these data are shown in Figure 3.55. Blood pH was lower (more acidic) for males 15 minutes before dosing, and 15 minutes after dosing in newborns.  118  Figure 3.55 Blood pH of newborn male and female lambs (AVG ± SEM).  119  4. Discussion	
   This thesis presents an in vivo evaluation of fluoxetine disposition in newborns up to ~1 year of age in chronically instrumented lambs. These studies revealed several findings: 1) the development of fluoxetine disposition is not a linear process; 2) the disposition of fluoxetine is stereoselective; 3) the binding of fluoxetine and norfluoxetine to plasma proteins and the plasma proteins levels increased as the lambs aged; and 4) there is a compelling sex difference in fluoxetine disposition.  4.1 Ontogeny	
  of	
  fluoxetine	
  disposition	
   Dissertation work by Dr. John Kim in our group investigated fetal-maternal, and adult non-pregnant sheep fluoxetine disposition; these data are summarized in Table 4.1 and Figure 4.1 and were compared with newborn values obtained in the studies described in this thesis. (Kim et al. 2004) To study fetal-maternal disposition, Dr. Kim administered an i.v. bolus dose of racemic fluoxetine to the fetus and pregnant-ewe on separate occasions.  Table 4.1 Fluoxetine pharmacokinetic parameters after i.v. bolus administration of racemic fluoxetine in fetal, pregnant, newborn and adult non-pregnant sheep (AVG ± SD). Pharmacokinetic Parameter  Fetal  Newborn  Maternal  Adult non-pregnant  Cl (ml/min/kg)  122 ± 33.8a  41.0 ± 10.1c  28.4 ± 8.25c  t1/2 (hr) Vdss (l/kg)  10.1 ± 0.80a 28.1 ± 13.2a  6.88 ± 2.20b 46.10 ± 12.9b 23.25 ± 11.29a  6.65 ± 1.6c 7.8 ± 1.2b  17.5 ± 5.5d 15.1 ± 3.8c  120  Data taken from the PhD thesis of Dr. John Kim (Kim 2004) The different letter superscripts indicate statistically significant values (1 way ANOVA)  Figure 4.1 Comparison of fluoxetine plasma pharmacokinetics between fetal, newborn, maternal, and adult non-pregnant sheep after i.v. bolus administration of racemic fluoxetine for clearance (a), elimination half-life (b), and volume of distribution at steady state (c). Data taken from the PhD thesis of Dr. John Kim.(Kim 2004)  The clearance of FX in the newborn (6.88 ± 2.20 ml/min/kg) was much lower than the fetus (122 ± 33.8 ml/min/kg), mother (41.0 ± 10.1 ml/min/kg), and adult non-pregnant (28.4 ± 8.25 ml/min/kg) sheep. Interestingly, Dr. Kim observed that fluoxetine clearance was greater during pregnancy using Student’s unpaired t-test in sheep. (Kim et al. 2004) This is similar to findings reported in humans (Heikkinen et al. 2003; Kim et al. 2006), and has been attributed in part at least to increased CYP2D6 activity in pregnancy (Wadelius et al. 1997).  In Dr. Kim’s sheep studies, fetal and maternal placental clearance values were calculated using equations described by Dr. Yeleswaram (Yeleswaram et al. 1993) Maternal placental clearance (CLmp) was calculated by multiplying fetal FX clearance by the fetal to maternal AUC ratio:  121  Fetal sheep placental clearance CLfp was calculated by multiplying maternal FX clearance by the ratio of maternal to fetal AUC:  Maternal sheep non-placental clearance (CLmn) was calculated by subtracting maternal FX placental clearance (CLmp) from maternal FX clearance (CLm):  Similarly, fetal sheep non-placental clearance (CLfn) was calculated by subtracting fetal FX placental clearance (CLfp) from fetal FX clearance (CLf):  Table 4.2 Fetal and maternal sheep placental and non-placental clearance values after i.v. bolus administration of racemic fluoxetine in separate studies to the fetus and mother. Data taken from the PhD thesis of Dr. John Kim.(Kim et al. 200) (AVG ± SD) CLmp (ml/min/kg) CLmn (ml/min/kg) CLfp (ml/min/kg) CLfn (ml/min/kg)  69.8 ± 22.7 38.0 ± 7.95 110.75 ± 49.3 11.1 ± 42.3  Fetal and maternal sheep placental and non-placental clearance values from Dr. Kim’s studies are summarized in Table 4.2. The calculated fetal non-placental clearance (Clfn) 11.1 ± 42.3 ml/min/kg was not significantly different from zero. Furthermore, Dr. 122  Kim’s studies did not observe Phase I and II metabolic pathways based on metabolite measurements in biological fluids, and in vitro studies with fetal hepatic microsomes. This led to the conclusion that the sheep fetus has little or no ability to metabolize FX on its own, and disposition was dependent on maternal trans-placental routes.  Interestingly, the arithmetic average of the sheep fetal non-placental clearance from Dr. Kim’s studies, 11.1 ± 42.3 ml/min/kg, was similar to FX clearance in sheep newborns, 6.88 ± 2.20 ml/min/kg. There is rapid and dynamic fetal growth during late gestation period. The large variance in the sheep fetal non-placental clearance may be due to variances in the development of drug disposition ability. The studies in pregnant sheep conducted by Dr. Kim were conducted between 124 and 137 d gestation (term ~ 147 d), whereas the Cohort 1 lambs had an average age of ~ 5 d. Further studies at ages between these 2 intervals may provide information on the development of drug disposition abilities in the sheep fetus.  Tuan Ahn Nguygen’s dissertation work studied the drug levels in postnatal lambs after in utero exposure to daily maternal FX dosing over the last 2 weeks of pregnancy. Figure 4.2 illustrates the drug level in postnatal lambs as a function of the time (in hours) since the last maternal dose. There is a gradual decline in drug levels as a function of postnatal age, which suggests the presence of drug metabolism ability. However, as indicated in Fig. 4.2, NFX was not detected in any of the lamb plasma samples, although it was present in the maternal blood samples collected at delivery (data not shown). The lack of detectable NFX in the lamb samples suggests that the FX elimination pathways at  123  this age do not involve FX demethylation and that this process must develop between birth and postnatal days 4-5 (i.e. cohort 1).  Figure 4.2 Plasma fluoxetine concentration in postnatal lambs as a function of the time since the last maternal FX dose after in utero exposure to chronic maternal fluoxetine dosing during pregnancy. Data taken from unpublished dissertation work by Tuan Anh Nguyen.  The volume of distribution of fluoxetine in fetal, newborn, maternal, and adult sheep are also summarized in Table 4.1 and Figure 4.1. Interestingly, values were comparable between the fetus (28.1 l/kg) and newborn (23.2 l/kg). In utero, the fetus has access to maternal trans-placental routes where the drug could distribute and this could result in a large volume of distribution for drugs in the fetus; however, it is interesting to note that fluoxetine Vdss is comparable between the fetus and newborn. Previous studies in our group observed a decrease in Vdss for valproic acid (H. Wong 2000) and diphenhydramine (Yeung 2004) in the postnatal lamb compared to the fetus, and this was hypothesized to be due to the lack of drug distribution into maternal compartments after  124  birth. The binding of valproic acid in the fetus (Kumar et al. 2000) and newborn (H. Wong et al. 2000) is ~50-60%.. The binding of diphenhydramine in the fetus is around 60%, and newborn is around 70%. (Yeung 2004) Fluoxetine is more highly bound than these drugs in the fetus (Kim 2004) and newborn (See section 3.1.5) at around 80-90%. Based on these data, the lower binding of valproic acid and diphenhydramine in utero resulted in a greater unbound fraction that distributed into maternal compartments and this may have caused the decrease in drug distribution observed postnatally. Conversely, the greater binding of fluoxetine to plasma proteins in utero resulted in a lower fraction that distributed into maternal compartments, and this may explain the comparable fluoxetine Vdss in the fetus and newborn lambs.  As mentioned in the introduction, dissertation work by Manoja Pretheeban (M.Sc.) in our group cloned a number of Phase I and II genes in sheep and investigated the mRNA and protein levels (CYP enzymes only) of these enzymes in the sheep fetus following either saline or cortisol administration, as well as in untreated newborn lambs, and adult sheep.(Pretheeban et al. 2012) The mRNA levels of several CYP enzymes are summarized in Figure 4.3 and that of several UGT enzymes are summarized in Figure 4.4.  125  Figure 4.3 Relative hepatic mRNA orthologue levels of CYP2C19, CYP2D6, and HNF4a in fetuses treated with saline or cortisol as well as in newborns, and adult sheep. Data taken from dissertation work by Manoja Pretheeban. (Pretheeban et al. 2012)  In general, these mRNA CYP2C19, CYP2D6, CYP2A6, and HNF4α levels increased as a function of age. Of relevance to this current study, levels are significantly greater in the adult sheep than in the newborn lamb. This observation is consistent with the current study in that the metabolism of FX increased as a function of age as the lambs aged from Cohorts 1 to 6. Also, the mRNA levels of CYP2C19 and CYP2D6 (enzymes involved in the demethylation of FX) are higher in newborns than in the late gestation fetus. This is consistent with the formation of measureable NFX levels in some of the Cohort 1 lambs. A similar developmental trend was observed for the UGT isozymes, which corroborates with the current study in that the metabolism of FX to Phase II glucuronide conjugates increased as a function of age. However, although there is an increase in the mRNA levels of the UGTs in newborn lambs, as discussed below, the results in this thesis suggest that Phase II metabolism of FX does not develop until around 1 month of  126  age. In the study of sheep Phase II enzymes by Preetheban et al, the UGT antibodies employed did not cross-react with sheep proteins; thus, the developmental of UGT proteins in sheep is not known. (Pretheeban 2011)  Figure 4.4 Relative hepatic mRNA orthologue levels of UGT1A6(a), UGT1A9 (b), UGT 2B7 (c), and HNF4α. Measured values from each experimental model is presented as solid circles, mean value +/- SEM given by open diamonds, different letters indicate significant differences. (FS, fetuses treated with saline; FC fetuses treated with cortisol; NB, untreated newborn lambs; AD, untreated adult sheep) Data taken from dissertation work of Manoja Pretheeban. (Pretheeban 2011)  In the current thesis work, Phase I metabolism of fluoxetine to norfluoxetine was observed in newborn lambs. Nine out of twelve lambs in this cohort metabolized FX to NFX, and all metabolized FX to TFMP. The plasma NFX to FX ratio was low at 0.179 ± 0.152. In the lambs in which NFX was not detected, metabolism of FX to NFX may have 127  taken place, but the level of NFX may have been below the LOQ (1 ng/ml) of the assay. No Phase II metabolism of fluoxetine or norfluoxetine to their glucuronides was observed in the newborn lambs.  The disposition of fluoxetine in newborn lambs (Cohort 1) involved Phase I metabolism pathways to NFX and TFMP, and renal excretion. The systemic clearance at this age was low at 6.88 ± 2.20 ml/min/kg. The renal clearance of fluoxetine was also low at 0.157 ± 0.0517 ml/min/kg. The amount of unchanged parent compound, fluoxetine, recovered in urine (132.7 ± 8.25 ug ) was significantly greater than TFMP (41.2 ± 4.3 ug). These data suggest the metabolism of fluoxetine in the newborn lamb is low, that FX is mainly excreted unchanged in the urine, and that the renal elimination of both the parent compound and its NFX and TFMP metabolites are low.  Previous work in our group evaluated the stereoselective disposition of fluoxetine during human pregnancy and breastfeeding, and in 2 day, 6 days, and 11 months of age. (Kim et al. 2006) At delivery, there was a high correlation between human maternal and umbilical cord blood fluoxetine and norfluoxetine concentrations. Notably, for 2 day old human infants exposed to the drug in utero, fluoxetine and norfluoxetine plasma concentrations were comparable to that in umbilical cord blood at delivery; another human study also reported similar observations (Heikkinen et al. 2003). Plasma fluoxetine and norfluoxetine concentrations fell progressively as the human infants aged. At 5 days after birth, human infants who had in utero fluoxetine exposure had only a 46% decline in plasma drug concentrations.(Rampono et al. 2004) Interestingly, Dr. Kim  128  observed that in all these age groups the mean norfluoxetine concentration was greater than fluoxetine. In both human mother and fetus, the disposition of fluoxetine and norfluoxetine was stereoselective with S/R ratios being significantly greater than 1.  As the lambs in the current study aged from newborn (Cohorts 1) to ~12 months of age (Cohort 6), significant maturational changes in the disposition of fluoxetine were observed. Metabolite to parent drug ratio (NFX/FX) was plotted as a function of fluoxetine systemic clearance from newborn to 1 year of age in Figure 3.12. There is a positive correlation between NFX/FX ratio and fluoxetine systemic clearance – this suggests an increasing role of this metabolic pathway in the disposition of fluoxetine as the newborn lamb matured to 1 year of age.  The disposition of fluoxetine in newborns lambs (Cohort 1) and ~ 10 days old (Cohort 2) lambs were comparable and included Phase I metabolism to NFX and TFMP, and renal excretion. For ~ 1 month old lambs (Cohort 3), Phase II metabolic pathways had developed. Quantitatively, as shown in Figure 3.26 the parent compound was predominantly recovered in urine, followed by TFMP, NFX-glucuronide, FXglucuronide, and NFX.  For ~3 month old lambs (Cohort 4), a significant increase in the metabolic capacity for fluoxetine was observed in the lambs as shown by two-way ANOVA and piecewise regression analyses of plasma pharmacokinetic parameters. A corresponding increase in urine metabolite levels was observed. The parent compound fluoxetine was no  129  longer the dominant compound recovered in urine; quantitatively, NFX levels were highest followed by FX-glucuronide and NFX-glucuronide. At this age, Phase I metabolism to NFX was the most important elimination pathway for fluoxetine, followed by Phase II metabolism to FX and NFX glucuronides, and finally Phase I metabolism to TFMP. As the lambs matured to ~6 months (Cohort 5), and ~12 months (Cohort 6), similar trends were observed in the disposition of fluoxetine – Phase I metabolism to NFX was the most important pathway for fluoxetine elimination, followed by Phase II metabolism to FX and NFX glucuronides, and then Phase I metabolism to TFMP. Renal excretion of FX and its metabolites was low. This is consistent with past maternal acute (Kim 2004) and chronic (Chien) sheep disposition studies in our group where renal excretion was a minor pathway for FX elimination.  In the current sheep studies following fluoxetine drug disposition from newborn to ~ 1 year of age, the most significant maturation changes were observed at ~1 month (Cohort 3) and ~ 3 months (Cohort 4). In addition to the changes in the metabolic pathways described previously, significant increases in the total plasma protein level, AAG, and drug protein binding were also observed at these ages. Cohorts #3 and #4 coincided with the period of time the lambs were weaned. The lambs were first observed to consume solid foods at 29.6 ± 2.2 days. (Tuan-Anh Nguyen’s unpublished data) The change from nutrients supplied from milk to solid foods in ruminant species is accompanied by anatomical, physiological and biochemical developmental changes in the body. (Baldwin et al. 2004) The literature in this area has focused primarily on intestinal growth and rumen development at the time of weaning.  130  (Shirazi-Beechey et al. 1991; Ruckebusch et al. 1983; Theriez et al. 1981) It would, however, be logical to surmise that development changes to other internal organs - such as the liver and kidney that are involved in drug disposition -undergo changes as well. A paper by Lane et al. reported on the hepatic development of ketogenic enzymes in lambs. Enzymes levels involved in hepatic ketogenesis were low before 42 days of age and a marked increase was observed by 42 days of age. (Lane et al. 2002) This corroborates with the period of time where significant maturational changes were observed in this study, and suggests that the expression of hepatic drug metabolizing enzymes may also increase at this time.  Immediately post parturition and during weaning, the sheep offspring must make anatomical changes to ensure survival by adapting to an external environment very different from the uterine environment, and adapting to a food source of solid foods from a liquid diet, respectively. During these periods, there is a marked accelerated gain in body weight, and differential growth of internal structures to best adapt to the environment to ensure survival. (Butterfield 1988) In Cohort #6, the lambs had an average age of 404.13 ± 24.2 days, and weight of 57.2 ± 6.78 kg. Dr. Kim’s dissertation work also included the disposition of FX after intravenous administration in adult non-pregnant ewes with an average body weight of 73.8 ± 11.6 kg.(Kim 2004) The body weights of Cohort #6 and the adult non-pregnant ewes studied in Dr. John Kim’s research were not significantly different based on Student’s t-test..  131  The adult non-pregnant ewes had a systemic clearance (CLs) of 28.4 ± 8.3 ml/min/kg, elimination half-life of 17.5 ± 5.5 hr, and volume of distribution of 15.1 ± 3.8 L/kg after FX intravenous administration.(Kim 2004) The non-pregnant ewes had a mean age of 1.8 ± 0.6 years. The female lambs in Cohort 6 (mean age = 1.1 ± 0.6 years) had a systemic clearance of 10.3 ± 2.8 ml/min/kg, elimination half-life of 8.6 ± 2.0 hrs, and volume of distribution of 5.03 ± 1.36 L/kg after intravenous administration. In general, FX in the female sheep of Cohort 6 had a lower clearance, but shorter elimination halflife and lower volume of distribution than the non-pregnant ewes. The relationship between these parameters is as follows, with adult non-pregnant ewe data shown on the left, and the corresponding Cohort 6 value shown on the right.  adult non-pregnant ewe  Cohort 6 female sheep  Compared to the adult non-pregnant ewes, the lower clearance and volume of distribution observed for Cohort 6 resulted in a lower elimination half-life. Nevertheless, these data suggest that there are further maturational changes in FX disposition and pharmacokinetics beyond 1 year of age in sheep.  132  4.2 Stereoselective	
  fluoxetine	
  and	
  metabolite	
  disposition	
   The disposition of FX was stereoselective in newborn lambs (Cohort 1). Plasma SFX concentrations were consistently greater than RFX, and the S/R ratio of the estimated pharmacokinetic parameters were consistently significantly different than a null hypothesis of 1. The systemic clearance of SFX (9.52 ± 3.01 ml/min/kg) was significantly lower than RFX (25.3 ± 7.17 ml/min/kg). Correspondingly, SFX has a greater volume of distribution (SFX 61.0 ± 25.2 L/kg, RFX 30.4 ± 5.8 L/kg), greater AUC (SFX 1964.2 ± 621.1 µg*hr/L, RFX 713.9 ± 204.9 µg*hr/L), greater MRT (SFX 76.76 ± 16.51 hr, RFX 43.9 ± 11.32 hr), and greater elimination half-life (56.9 ± 9.6 hr) than RFX (33.0 ± 10.7 hr) than RFX. The metabolism of FX to NFX was stereoselective. The S/R ratio of NFX/FX was significantly different than a null hypothesis of 1. The amount of SFX recovered in urine was significantly greater than RFX, as was the renal clearance of SFX versus RFX.  Plasma and urine PK data compellingly suggests that after intravenous FX administration and for the duration of the experiments, the level of the S isomer is consistently higher than R. The stereoselective disposition of FX has been hypothesized to be due to the following: differential enzyme interactions; and/or plasma protein and/or tissue binding. Based on our protein binding studies, the fraction unbound for SFX was less than RFX – more SFX is bound to plasma proteins, and thus prolonging its levels in the body. The S/R ratio is consistently significantly different than a null hypothesis of 1.  133  To further evaluate the contribution of drug plasma protein binding on the stereoselective disposition of fluoxetine, the free clearance of fluoxetine was calculated; these data are summarized in Table 3.7. As mentioned previously, it is important to note that only unbound drug in the body undergoes metabolism and elimination. Free fluoxetine clearance was calculated for Cohorts 1 to 6. The resulting free drug clearance values for R and S fluoxetine isomers are much closer compared with total drug clearance estimates resulting in an S/R ratio that is not different from 1 (1.02 ± 0.06). These data suggest that differential plasma protein binding of FX isomers is largely responsible for the stereoselective disposition of FX isomers after acute administration in postnatal lamb from newborn up to 1 year of age. This is consistent with the findings of Dr Kim (Kim 2004), who reported that in pregnant sheep given i.v. bolus injection of FX, the stereoselective disposition was largely due to stereoselective protein binding.  While stereoselective FX binding to plasma protein has been experimentally demonstrated in this study, other factors that may contribute to stereoselectivity may be possible. The distribution of a drug is dependent on tissue binding in addition to plasma protein binding. The contribution of tissue and protein binding is summarized by the following equation (Vp is the volume of distribution; Vb is the blood volume; Vt is the tissue volume; fb is the unbound fraction in blood; and ft is the unbound fraction in tissues):(Wilkinson 1983)  134  Unfortunately tissue-binding studies were not conducted in our studies so no conclusions can be drawn about the contribution of tissue binding to stereoselective disposition.  Dr. Kim’s dissertation work further investigated the in vitro stereoselective metabolism of FX to S- and RNFX using fetal and maternal sheep hepatic microsomes. (Kim 2000) These data are summarized in Table 4.3. Unfortunately, fetal sheep data were not available as NFX concentrations were below the LOQ of the assay. Adult S/R NFX ratios were very close to 1; however, variability data are not available. In addition, Table 4.4 summarizes the stereoselective formation NFX using cDNA expressed CYP isozyme microsomes.  Table 4.3 Stereoselective formation of norfluoxetine in adult and fetal sheep measured by in vitro microsome studies. Data taken from dissertation work of Dr. John Kim. (Kim 2000)  135  Table 4.4 Stereoselective formation of norfluoxetine using human cDNA-expressed CYP isozyme microsomes. Data taken from dissertation work by Dr. John Kim. (Kim 2000)  Contrary to the in vitro adult sheep microsome metabolism studies, individual cDNA preparations of CYP isozyme microsomes, specifically CYPS 2C9, 2C18, and 2C19, exhibited stereoselective metabolism of FX to NFX with the rate of RNFX formation being greater than for SNFX.  For a drug such as FX that is principally metabolized, the well-stirred model describes the relationship between protein binding, hepatic blood flow, and hepatic intrinsic clearance on hepatic drug clearance. These relationships are illustrated in the following equation where QH is the hepatic blood flow, ClH is the hepatic clearance, ClINT  136  is the intrinsic clearance of hepatocytes, and fub is the fraction of unbound drug in plasma: (Wilkinson and Shand 1975)  For low clearance drugs such as FX, the intrinsic clearance of hepatocytes is negligibly small and the denominator approximates the value of hepatic blood flow. Hepatic clearance then is a function of the fraction unbound and intrinsic clearance of hepatocytes:  Therefore, the stereoselective hepatic clearance of a drug is dependent on both protein binding and metabolism by hepatocytes. Both of these processes can be stereoselective and may then explain the results reported in this thesis.  Levels of FX and NFX glucuronides observed in the urine of the lambs of the current studies were not stereoselective. In vitro metabolism studies using cDNA UGT isozymes may provide information on the formation rates of S and R isomers of FX and NFX glucuronides and whether their ratios are significantly different than 1. There is a paucity of data in the literature regarding the stereoselective conjugation by UGTs. There are studies available but of structurally dissimilar compounds that suggest stereoselective conjugation of oxazepam by UGT2B7, UGT1A9 (Duan et al. 2002), and phenytoin by UGT 1A1, 1A9, and 2B15 (Nakajima et al. 2007). Likewise, plasma protein binding  137  studies would provide information on whether there is stereoselective binding of S and R FX and NFX glucuronides. However, plasma protein binding of these Phase II conjugates are not expected to be significant. The premise of Phase II metabolism is to increase the polarity and water solubility of the compound to facilitate excretion. Such compounds would not be expected to be highly bound to plasma proteins.  The stereoselective disposition of FX and its metabolites in sheep were comparable between sex, and developmentally there did not appear to be differences as the lambs aged from newborn (Cohort 1) to ~1 year of age (Cohort 6). Interestingly, the stereoselectivity of FX disposition from cohorts 1 to 6 is comparable to the fetus, and adult non-pregnant ewes.(Kim 2004) The mechanism(s) involved in stereoselectivity appears to be conserved from the fetus to the adult, with all of the data suggesting this is due to plasma protein binding based on stereoselective fluoxetine free clearance at least with acute dosing.  Chronic FX dosing studies in pregnant women (Kim et al. 2006), and pregnant sheep (Chien 1997) in our group indicated that stereoselective metabolism comes into play with chronic dosing. Human population studies of extensive and poor CYP2D6 metabolizers after single oral dose found significant differences between these metabolizers (Fjordside et al. 1999; Alfaro et al. 2000; Hamelin et al. 1996). In vitro studies have found that both FX and NFX are inhibitors of CYP2D6, with the S isomers being more potent inhibitors than the R isomer. (Stevens et al. 1993; Alfaro et al. 2000; Lam et al. 2002) Therefore, with chronic FX therapy the FX/NFX-mediated inhibition of  138  CYP2D6 would reduce the metabolism of SFX since this P450 isoform is primary responsible for its metabolism. (Ring et al. 2001) In contrast, the metabolism of RFX would be minimally affected since it is preferentially metabolized (compared to SFX) by CYP2C9/2C19 (Ring et al. 2001), so that its clearance is higher and serum levels lower compared to the S-isomer.  4.3 Fluoxetine	
  and	
  norfluoxetine	
  plasma	
  protein	
  binding	
  and	
  developmental	
   plasma	
  protein	
  levels.	
   In the current postnatal sheep studies, free plasma fractions of FX and NFX decreased significantly, and total plasma protein and AAG increased significantly as the lambs aged from newborn (Cohorts 1) to ~1 year of age (Cohort 6) (see section 3.1.6).  The increase in plasma AAG level as a function of age has been reported in several human studies in the literature. (Wood 1981; Davis 1985; Wallace 1976; Verbeeck 1984) This is best illustrated in a human study by Wood et. al. that measured AAG in the fetus, pregnant women, adult males and females, and also females on oral contraceptive. Fetal levels of AAG were significantly lower than in adult. These data are summarized in Figure 4.5. (Wood 1981; van der Sluijs et al. 1985) In the current study, as the lambs aged from Cohort #1 to #6, a similar trend of increasing plasma AAG concentration was observed; these data are provided in Figure 3.35.  139  Figure 4.5 AAG levels from study by Wood et. al. 1986 where increasing levels of alpha1-acid glycoprotein (AAG) in plasma were observed as a function of age. Data taken from published work by Wood et al. (Wood 1981)  Human studies reported in the literature suggest that sex differences play little role in contributing to alterations in plasma drug binding as neither albumin nor AAG levels reported in several studies are significantly different between sex. (Wallace 1976; Wood 1981; Pruitt et al. 1971; Bender et al. 1975; Wood 1986) Consistent with these findings, no sex differences were observed for plasma AAG levels in the current studies in postnatal lambs, as summarized in Figure 3.54.  Drug plasma protein binding can be influenced by blood pH. In humans, arterial blood pH values compatible with life are reported to range from 6.7 to 8.0. (SiggaardAndersen 1971; Velez et al. 1985; Harrisons 2008) Disruptions to the hydrogen homeostasis system and hence blood pH have either respiratory or metabolic origins, and may be caused by drugs. (Harrisons 2008) Drugs with extensive and pH-dependent 140  protein binding with a narrow-therapeutic-range and a high extraction ratio are of concern in terms of the pH effects.(Benet et al. 2002). FX is a highly protein bound drug, and has a low extraction ratio. Several studies in humans investigated the effect of pH on protein binding of basic compounds. Basic drugs demonstrate an inverse relationship with fub ; an increase in pH results in a decrease in fub. Regardless of whether basic drugs are bound to either albumin or AAG this pH sensitivity remains the same. (Urien et al. 1993; Hinderling et al.) In the current postnatal sheep studies, a significant sex difference was observed in blood pH between male (7.36 ± 0.0325) and females (7.42 ± 0.0220) in the Cohort 1 lambs up to 15 minutes after FX dosing, as summarized in section 3.2.4, Figure 3.38. Female lambs have a significantly higher pH, which would hypothetically result in lower fub. In contrast, a higher fub was observed for female lambs than male lambs in Cohort #1. Moreover, pH is dependent on temperature, and temperature has been reported to influence the extent of protein binding of drugs in human studies. (Paxton et al. 1983) Temperature data are summarized in Appendix 3. No significant differences in blood temperature were observed between sex in the current sheep studies; thus, this is unlikely to be the cause of the observed pH difference.  Alternatively, pH-induced changes in the fraction of ionized and non-ionized drug could be responsible for the observed pH-dependent interaction with plasma proteins. The non-ionized fraction of a basic drug that is increased at alkaline pH could preferentially bind to plasma protein. (Höllt 1975; McNamara et al. 1981; Hinderling et al. 2005; Otagiri et al. 1989) Nevertheless, the effects of blood pH and temperature on  141  protein binding observed in the current studies appear to be negligible. The increase in drug plasma protein binding as the lambs aged from newborn to ~1 year of age was concurrent to an increase in plasma AAG and total protein levels.  4.4 Sex	
  differences	
  in	
  fluoxetine	
  disposition	
  	
   In plasma, the clearance of FX in Cohort 1 was significantly greater in female lambs (8.67 ± 0.883 ml/min/kg) than male lambs (4.83 ± 0.568 ml/min/kg). AUC was 1934.6 ± 175.5 µg*hr/L for female lambs and was significantly lower than the 3313.9 ± 396.1 µg*hr/L value calculated for male lambs. Correspondingly, MRT was significantly shorter in female lambs (44.54 ± 4.34 hr) than male lambs (78.90 ± 15.44 hr). However, the metabolism of FX to NFX as measured by NFX AUC was not significantly different between sex. The NFX/FX ratio was 0.152 ± 0.13 for female lambs, and 0.099 ± 0.18 for male lambs. This lack of a significant difference may have resulted in part from the use of truncated AUC estimates as plasma levels of norfluoxetine had not rapidly declined by the end of the experiment. The weight-adjusted renal clearance of FX was significantly greater for female lambs (0.265 ± 0.0623 ml/min/kg) than male lambs (0.126 ± 0.0408 ml/min/kg). Moreover, the amount of FX measured in urine was significantly greater for female lambs (151.8 ± 15.95 µg) than male lambs (113.9 ± 10.6 µg). Renal accumulation of TFMP was comparable between sex: male lambs were 72.5 ± 9.31 µg and female lambs were 79.2 ± 7.80 µg, though this value may have been underestimated as the data suggest that renal excretion of this metabolite continued after the experiment. Overall, the data suggest that newborn female lambs were more able to metabolize and eliminate fluoxetine than male lambs. Moreover, piecewise regression analysis (section 3.2) 142  suggested sex differences beyond newborns and throughout the first year of life where developmental changes in FX Cl were more rapid in male than female lambs. Analysis of the female-male lamb differences in the mean clearance values in each cohort via the paired Student’s t-test confirmed that over the age range studied, FX clearance was higher in female than male lambs.  The plasma protein binding of FX was greater for male than female lambs, though this difference was not significantly different based on two-way ANOVA. The fraction unbound was 12.1 ± 1.66 % in female lambs, and 10.0 ± 1.40 % in male lambs. Likewise, the plasma protein binding of NFX, though greater in male than female lambs, was not significantly different based on two-way ANOVA between sex; the fraction unbound was 11.8 ± 1.06 in female lambs, and 9.6 ± 1.54 in male lambs. Neither the total protein nor AAG levels in plasma, though greater in male lambs, were significantly different between sex based on two-way ANOVA. The neonatal/newborn period is one of rapid and dynamic growth. As the two-way ANOVA statistical analysis calculates the average based on age and sex groups, potential individual differences in development during this period may have been missed.  To further evaluate the potential contribution of drug plasma protein binding on the sex difference that was observed, the free clearance of fluoxetine was calculated, as shown in Table 3.12. The calculated free clearance for fluoxetine was larger for females (86.13 ml/min/kg) than males (40.30 ml/min/kg) for newborn lambs. Free fluoxetine clearance does not appear to mitigate the observed sex difference in fluoxetine clearance.  143  The volume of distribution of a drug is dependent on both protein and tissue binding. The relationship between volume of distribution, protein binding, and tissue binding can be described as follows where Vb is the blood volume, Vt volume of tissues, fT is the unbound drug in tissue, and fb is the unbound drug in blood.(Wilkinson 1983)  Due to the unavailability of tissue binding data, the significance of tissue binding could not be determined. However, the volume of distribution of FX is comparable between male (23.40 ± 4.50 L/kg) and females (23.16 ± 16.13 L/kg) lambs for this cohort. Thus, the impact of a sex difference in tissue drug binding appears to be minimal.  The relationship between elimination half-life, volume of distribution, and systemic clearance could be summarized by the following formula, though it is important to note that a drug’s volume of distribution and systemic clearance are independent physiologically:  Substituting in the values for females (shown on the left) and female lambs (shown on the right) for male lambs (Cohort 1), the following mathematical relationship is obtained:  144  female lambs  male lambs  It can be observed that a greater systemic clearance in female lambs resulted in a shorter elimination half-life relative to male lambs. Hepatic and renal elimination mechanisms are the two major clearance pathways in the developing infant.(Alcorn et al. 2002) These data suggest that female lambs may have greater levels of hepatic drug metabolizing enzymes resulting in greater hepatic elimination. Even though the experimentally measured NFX/FX ratios (0.152 ± 0.13 for females, and 0.099 ± 0.18 for males) were not statistically significantly, the variances were quite high – this may perhaps be due to NFX being present at such low levels and truncated AUC estimation being used. Nevertheless, the levels of hepatic mRNA and enzymes warrant investigation between sex in this cohort.  Phenotypic sex differences are obvious for most mammal species. In 1916, Lillie suggested the influence of chemical factors in sex determination in his research whereby mixing the placental blood supply of male and female calves produced a “more masculine” female. Since Lillie’s research, there has been a plethora of research in the literature investigating hormonal, chemical, and anatomical differences between male and female sexes. (Rinn et al. 2004) However, there is a paucity of information on the specific differences between male and female that gives rise to sexual dimorphism, especially from a developmental viewpoint in regards to drug disposition.  145  In rodent studies, males were more susceptible to impaired renal function due to a direct reduction of the number of nephrons (Woods et al. 2004), and decreased glomerular filtration rate and papillary volume(Saez et al. 2007). Similar to rodent data, studies in sheep have also demonstrated reduced glomerular filtration rate in male offspring from prenatal steroid exposure(Tang et al. 2009), and nephrogenesis impairment in male offspring exposed to nutrient restriction. (Gilbert et al. 2007; Aiken et al. 2012)  A recent study investigated the gene expression differences in male and female adult mice hypothalamus, kidney, liver, and reproductive tissues. Significant differences in gene expression were observed in the kidney, liver, and reproductive tissues. The majority of genes differentially expressed in the kidney and liver are involved in drug and steroid metabolism and osmotic regulation. This study raises important implications for differential drug disposition abilities for male and female mammals. (Rinn et al. 2004)  A plethora of data supports that dimorphic patterns of growth-hormone release regulates sex specific gene expression in the liver. In rodent and human studies, both males and females were observed to have cyclical release of growth hormones, but females have smaller amplitudes and more frequent pulses, resulting in almost continuous plasma levels of growth hormones.(Agrawal et al. 2000) Different growth-hormone profiles between adult sexes can affect the expression of numerous genes. (Rinn et al. 2005) Transcription factors, such as HNF4α, have recently been suggested to regulate  146  gene expression in a sex-specific manner in mice liver. This study demonstrated that HNF4α-deficient mice lose sex-related expression of several CYP450s. (Wiwi et al. 2004) Another recent study identified other transcription factors in the mouse liver that regulate sex-specific transcription independent of hormones.(Krebs et al. 2009) These studies suggests the possibility of sex-specific gene expression by a complex transcriptional circuitry that could be influenced by sex hormones. (Rinn et al. 2005)  The sex difference observed in the current postnatal sheep studies was greatest in newborns (Cohort 1). A study by Savoie et. al. suggested that a surge in testosterone level in male lambs do not occur until approximately 3 days of age. (Savoie et al. 1981) Yarney et al reported the onset of male lamb puberty to occur between 150 to 200 days (~28 weeks ) of age with a significant increase in testosterone level. (Yarney et al. 1989) Suttie et al reported the onset of female lamb puberty to occur at ~30 weeks with a significant increase in progesterone level.(Suttie et al. 1991) Hypothetically, the delay in newborn male testosterone level surge, and subsequent comparable onset of puberty between sex in sheep may offer an explanation of why for all the age groups the greatest sex difference was observed in the newborn (Cohort 1).  In a study of experimentally induced moderate premature birth in sheep, a sex difference in the survival of preterm lambs was observed, with the viability of male lambs significantly lower than female lambs was reported by De Matteo et al. The rate of death for male lambs was 2.75 times greater than female lambs. De Matteo et. al. hypothesized that the male lambs died from respiratory insufficiency - either the lungs or  147  neural control of breathing is less well developed in preterm males than females.(Stokes et al. 2010) Analogously, perhaps there was a similar lack in maturity in drug disposition organs in the term lambs in Cohort #1. There is an abundance of data in the human population on the greater mortality in human males compared to females born prematurely.  Recently, De Mattero et. al. published their study on sex differences in cardiorespiratory transition and lung surfactant composition following premature birth in sheep to identify the mechanisms involved for the greater respiratory morbidity and mortality for male preterms. (Ishak et al. 2012) Their studies suggested no sex differences in lung architecture, but differences in surfactant phospholipid composition and function which may compromise gas exchange and impair respiratory adaptation. Moreover, their studies reported significantly lower blood pH, which was also observed in our studies.  From an evolutionary biology viewpoint, sexual dimorphism has been postulated to be due to species survival in the Trivers-Willard hypothesis (Trivers and Willard 1973). Sexual dimorphism could be regarded in terms of energy-expenditure investment by the mother. Nourishment in offspring somatic tissues are costly energy-investments that would be made preferentially to female offspring whose overall physiology and reproductive tracts must be protected to ensure their reproductive abilities to propagate the species. Conversely, for the male offspring only germ cells must be protected to be fit to mate. (Aiken et al. 2012)  148  4.5 	
  Conclusions	
   Newborn lambs were able to eliminate fluoxetine by processes that included Phase I pathways to NFX and TFMP, and renal elimination. However, the overall disposition of fluoxetine in the newborn lamb was limited compared to the adult sheep. The development of fluoxetine disposition ability from newborn to ~1 year of age in sheep was not a linear process with time. Rather, a significant increase in FX elimination was observed at ~3 months, which was the result of significant maturation of Phase I and Phase II pathways, and renal parent drug and metabolite elimination.  These studies present data against the assumption that growth in the pediatric population is a linear process, which is the premise of allometric scaling for pediatric pharmacokinetics. It may be argued that further research is needed in the area of pediatric pharmacokinetics, though the challenges of performing pharmacokinetic experiments in the human pediatric population that had stymied research in this area continues to be present. Alternative approaches such as population modeling are promising.  The disposition of fluoxetine in sheep was stereoselective from newborn to ~1 year of age. This stereoselectivity appeared to be comparable to the fetus and adult sheep. The similarity of free clearance calculated for fluoxetine enantiomers compellingly suggested that stereoselective plasma protein binding was the main mechanism for the stereoselective fluoxetine disposition.  149  The gender sex difference in fluoxetine disposition in sheep warrants further investigation. In vivo pharmacokinetic data implicated systemic clearance to be significantly different between male and female newborn lambs, and that the development of fluoxetine systemic clearance was more rapid in male lambs than female lambs in the first year of life. Studies on sex differences in hepatic enzyme expression and renal development in the literature were discussed. Further work to examine sex differences in hepatic enzyme expression and kidney function in sheep may provide mechanistic information on this sex differences in drug disposition.  The in vivo sheep studies in this thesis are limited by the lack of in vitro studies to provide mechanistic information for these observations. In particular, hepatic drug metabolizing enzyme levels between newborns to ~1 year of age could provide significant information regarding the sex difference and developmental maturation observed in these in vivo studies. Further, in vitro studies to evaluate the influence of growth hormones on HNF4α and the expression of drug metabolizing enzymes could provide insights into such mechanisms in a large animal species. Moreover, in vitro evaluation of stereoselective fluoxetine UGT conjugation would add data to the paucity of information on UGTs in the literature.  150  Bibliography	
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The University of British Columbia. Wood, A.J.J., G.L. Kearns, S.M. Abdel-Rahman, S.W. Alander, D.L. Blowey, J.S. Leeder, and R.E. Kauffman. 2003. “Developmental Pharmacology—Drug Disposition, Action, and Therapy in Infants and Children.” New England Journal of Medicine 349 (12): 1157–1167. Wood, M. 1986. “Plasma Drug Binding.” Anesthesia & Analgesia 65 (7): 786–804. Wood, M., and A.J.J. Wood. 1981. “Changes in Plasma Drug Binding and Α1-Acid Glycoprotein in Mother and Newborn Infant.” Clinical Pharmacology & Therapeutics 29 (4): 522–526. Woods, Lori L, Douglas A Weeks, and Ruth Rasch. 2004. “Programming of Adult Blood Pressure by Maternal Protein Restriction: Role of Nephrogenesis..” Kidney International 65 (4): 1339–1348.  172  Yarney, T A, and L M Sanford. 1989. “Pubertal Changes in the Secretion of Gonadotropic Hormones, Testicular Gonadotropic Receptors and Testicular Function in the Ram.” Domestic Animal Endocrinology 6 (3): 219–229. Yeleswaram, K, D W Rurak, E Kwan, C Hall, A Doroudian, M R Wright, F S Abbott, and J E Axelson. 1993. “Transplacental and Nonplacental Clearances, Metabolism and Pharmacodynamics of Labetalol in the Fetal Lamb After Direct Intravenous Administration..” The Journal of Pharmacology and Experimental Therapeutics 267 (1): 425–431. Yeung, Sam Au. 2003. “CNS Pharmacokinetics of Dipengydramine in Sheep” PhD diss. The University of British Columbia. Yoo, S D, D W Rurak, S M Taylor, and J E Axelson. 1993. “Transplacental and Nonplacental Clearances of Diphenhydramine in the Chronically Instrumented Pregnant Sheep..” Journal of Pharmaceutical Sciences 82 (2): 145–149. Zajecka, J M, R Weisler, and G Sachs. 2002. “A Comparison of the Efficacy, Safety, and Tolerability of Divalproex Sodium and Olanzapine in the Treatment of Bipolar Disorder.”Journal Of Clinical Psychiatry. 63:1148-1155. 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   Appendices	
   Appendix	
  1	
  Plasma	
  fluoxetine	
  pharmacokinetic	
  data	
  of	
  individual	
  lambs	
   1.1 Lambs in Cohort #1 L740M - Dose: 4.9mg - Age: 2 days PK Parameter RFX SFX FX AUC (µg*hr/L) 656.6 2776.9 FX Elimination Half-life (hr) 79.7 215.3 FX Volume of Distribution Steady 89.9 157.6 State (L/kg) FX Clearance (ml/min/kg) 12.7 3.01 FX Mean Residence Time (hr) 101.9 281.6 NFX AUC (µg*hr/L) 151.7 208.5 L741M - Dose: 5.1mg - Age 3 days PK Parameter RFX SFX AUC (µg*hr/L) 847.8 2420.9 Elimination Half-life (hr) 29.9 46.9 Volume of Distribution Steady 27.3 48.8 State (L/kg) Clearance (ml/min/kg) 9.83 3.50 Mean Residence Time (hr) 41.4 66.1 NFX AUC (µg*hr/L) 240.3 342.8 L310DF - Dose: 4.1 mg - Age 2 days PK Parameter RFX SFX AUC (µg*hr/L) 1009.4 1834.7 Elimination Half-life (hr) 26.4 34.8 Volume of Distribution Steady 25.9 37.1 State (L/kg) Clearance (ml/min/kg) 8.17 4.67 Mean Residence Time (hr) 37.5 47.6 NFX AUC (µg*hr/L) L82M - Dose: 5.9 mg - Age 6 days PK Parameter RFX SFX AUC (µg*hr/L) 924 2795.5 Elimination Half-life (hr) 32.7 42.8 Volume of Distribution Steady 21.7 49.7 State (L/kg) Clearance (ml/min/kg) 9.00 2.83 Mean Residence Time (hr) 45.3 60.8 NFX AUC (µg*hr/L) 172.6 203.8  S/R 4.23 2.70  FX 3433.3 148.8  1.75  56.0  0.24 2.76 1.37  4.83 191.2 360.2  S/R 2.86 1.57  FX 3178.7 39.6  1.79  17.5  0.35 1.60 1.43  5.17 55.5 583.1  S/R 1.82 1.32  FX 2862.8 33.6  1.43  15.6  0.56 1.27 -  5.83 44.8 -  S/R 3.03 1.31  FX 3658.6 38.8  2.29  15.1  0.32 1.34 1.18  4.50 55.1 376.5  174  L620M - Dose: 5.5 mg - Age 6 days PK Parameter RFX SFX AUC (µg*hr/L) 950.36 2696.13 Elimination Half-life (hr) 33.98 55.02 Volume of Distribution 28.17 47.32 Steady State (L/kg) Clearance (ml/min/kg) 8.75 3.00 Mean Residence Time (hr) 44.97 75.94 NFX AUC (µg*hr/L) 63.9 92.0 L371M - Dose: 6.8 mg - 5 days PK Parameter RFX SFX AUC (µg*hr/L) 950.28 2797.64 Elimination Half-life (hr) 37.44 52.16 Volume of Distribution Steady 46.30 25.10 State (L/kg) Clearance (ml/min/kg) 8.76 3.03 Mean Residence Time (hr) 43.99 70.21 NFX AUC (µg*hr/L) 215.8 319.1 L77F1 - Dose: 6.1 mg - Age 5 days PK Parameter RFX SFX AUC (µg*hr/L) 468.2 1484.5 Elimination Half-life (hr) 24.0 37.2 Volume of Distribution Steady 33.8 70.7 State (L/kg) Clearance (ml/min/kg) 18.3 5.51 Mean Residence Time (hr) 33.1 50.1 NFX AUC (µg*hr/L) L740F - Dose: 3.3 mg - Age 3 days PK Parameter RFX SFX AUC (µg*hr/L) 513.4 1635.2 Elimination Half-life (hr) 17.5 42.9 Volume of Distribution Steady 35.9 48.5 State (L/kg) Clearance (ml/min/kg) 15.8 5.33 Mean Residence Time (hr) 24.9 58.7 NFX AUC (µg*hr/L) PRE03F1 - Dose: 6.6 mg - Age 4 days PK Parameter RFX SFX AUC (µg*hr/L) 549.9 1386.8 Elimination Half-life (hr) 27.4 30.3 Volume of Distribution Steady 29.6 67.3 State (L/kg) Clearance (ml/min/kg) 15.2 5.99 Mean Residence Time (hr) 37.0 41.1 NFX AUC (µg*hr/L) 286.6 299.2 L741F - Dose: 5.0 mg - Age 3 days  S/R 2.84 1.62  FX 3547.3 46.18  1.68  17.83  0.35 1.69 1.44  4.67 63.25 155.9  S/R 2.94 1.39  FX 3759.27 49.19  0.54  16.91  0.34 1.60 1.36  4.49 63.55 589.1  S/R 3.17 1.55  FX 1874.9 32.5  2.09  24.1  0.32 1.51 -  8.33 45.1 -  S/R 3.19 2.45  FX 2088.6 33.7  1.35  21.9  0.32 2.36 -  8.03 45.7 -  S/R 2.52 1.11  FX 1950.2 29.5  2.27  20.3  0.40 1.11 1.06  8.52 39.6 585.8  175  PK Parameter RFX SFX AUC (µg*hr/L) 561.9 1092.3 Elimination Half-life (hr) 34.5 55.4 Volume of Distribution 42.6 130.1 Steady State (L/kg) Clearance (ml/min/kg) 14.8 7.67 Mean Residence Time (hr) 46.6 73.1 NFX AUC (µg*hr/L) 156.0 172.8 L752F1 - Dose: 5.9 mg - Age 5 days PK Parameter RFX SFX AUC (µg*hr/L) 532.50 1435.79 Elimination Half-life (hr) 27.30 34.16 Volume of Distribution Steady 33.02 67.44 State (L/kg) Clearance (ml/min/kg) 15.7 5.83 Mean Residence Time (hr) 35.91 47.41 NFX AUC (µg*hr/L) 109.6 149.3 L752F2 - Dose: 5.0 mg - Age 5 days PK Parameter RFX SFX AUC (µg*hr/L) 591.83 1557.7 Elimination Half-life (hr) 25.20 35.48 Volume of Distribution Steady 31.09 57.76 State (L/kg) Clearance (ml/min/kg) 14.2 5.33 Mean Residence Time (hr) 34.18 48.43 NFX AUC (µg*hr/L) 286.6 299.2  S/R 1.94 1.61  FX 1624.9 38.9  3.05  32.1  0.52 1.57 1.11  10.3 52.1 328.9  S/R 2.70 1.25  FX 1961.42 30.60  2.04  21.17  0.37 1.32 1.36  8.49 41.52 258.8  S/R 2.63 1.41  FX 2107.38 31.41  1.86  20.51  0.38 1.42 1.04  7.83 43.22 585.8  1.2 Lambs in Cohort #2 L741M - Dose: 7.1 mg - Age 11 days PK Parameters RFX SFX FX AUC (µg*hr/L) 999.4 3641.5 FX Elimination Half-life (hr) 30.2 43.7 FX Volume of Distribution Steady 17.0 40.54 State (L/kg) FX Clearance (ml/min/kg) 8.32 2.17 FX Mean Residence Time (hr) 40.5 61.9 NFX AUC (µg*hr/L) 560.5 727.3 L740M - Dose: 7.4mg - Age 12 days PK Parameters RFX SFX FX AUC (µg*hr/L) 1028.7 1923.1 FX Elimination Half-life (hr) 45.04 72.08 FX Volume of Distribution Steady 31.79 96.49 State (L/kg) FX Clearance (ml/min/kg) 7.99 4.33 FX Mean Residence Time (hr) 61.1 99.3 NFX AUC (µg*hr/L) 719.1 713.8  S/R 3.644 1.45  FX 4557.4 38.9  2.38  11.9  0.274 1.53 1.29  3.65 54.6 1287.8  S/R 1.87 1.60  FX 2884.0 50.74  3.04  177.35  0.53 1.62 1.02  5.83 69.1 1432.9 176  L310DF - Dose: 7.4 mg - Age 12 days PK Parameters RFX SFX FX AUC (µg*hr/L) 635.4 3188.1 FX Elimination Half-life (hr) 41.3 56.3 FX Volume of Distribution Steady 24.7 87.9 State (L/kg) FX Clearance (ml/min/kg) 13.2 2.49 FX Mean Residence Time (hr) 55.88 78.8 NFX AUC (µg*hr/L) 453.5 518.1 L52M - Dose: 7.3 mg - Age 12 days PK Parameters RFX SFX FX AUC (µg*hr/L) 1756.7 3238.9 FX Elimination Half-life (hr) 40.12 46.06 FX Volume of Distribution Steady 20.20 31.65 State (L/kg) FX Clearance (ml/min/kg) 4.75 2.58 FX Mean Residence Time (hr) 55.60 65.42 NFX AUC (µg*hr/L) 243.9 748.8 L645M1 - Dose: 8.6 mg - Age 12 days PK Parameters RFX SFX FX AUC (µg*hr/L) 437.9 1195.5 FX Elimination Half-life (hr) 37.7484 41.62141 FX Volume of Distribution 17.9 42.6 Steady State (L/kg) FX Clearance (ml/min/kg) 9.49 2.52 FX Mean Residence Time (hr) 50.9 58.4 NFX AUC (µg*hr/L) 570.9 643.1 L620M - Dose: 7.8 mg - Age 13 days PK Parameters RFX SFX FX AUC (µg*hr/L) 1082.6 3029.6 FX Elimination Half-life (hr) 27.6 34.8 FX Volume of Distribution 16.5 33.9 Steady State (L/kg) FX Clearance (ml/min/kg) 7.67 2.67 FX Mean Residence Time (hr) 36.7 49.9 NFX AUC (µg*hr/L) 287.6 643.1 L752F - Dose: 12.8 mg - Age 20 days PK Parameters RFX SFX FX AUC (µg*hr/L) 384.22 536.63 FX Elimination Half-life (hr) 10.328 24.7 FX Volume of Distribution 19.0 66.9 Steady State (L/kg) FX Clearance (ml/min/kg) 21.7 15.5 FX Mean Residence Time (hr) 10.2 25.7 NFX AUC (µg*hr/L) 239.3 297.6  S/R 5.02 1.36  FX 3795.0 51.9  3.56  18.9  0.20 1.41 1.14  4.42 71.73 971.8  S/R 1.84 1.15  FX 4951.8 42.94  1.57  12.24  0.54 1.18 3.07  3.33 60.60 992.7  S/R 2.73 1.10  FX 2258.2 41.6  2.38  17.9  0.37 1.15 1.12  7.28 58.4 1220.6  S/R 2.80 1.26  FX 3828.4 40.2  2.06  13.7  0.36 1.36 1.04  4.35 52.5 1220.6  S/R 1.40 2.39  FX 950.66 12.67  3.51  11.8  0.72 2.52 1.24  17.5 11.2 536.9  177  L740F - Dose: 5.1 mg - Age 14 days PK Parameters RFX SFX FX AUC (µg*hr/L) 1307.07 2704.29 FX Elimination Half-life (hr) 30.78 48.48 FX Volume of Distribution 21.5 52.6 Steady State (L/kg) FX Clearance (ml/min/kg) 6.33 2.99 FX Mean Residence Time (hr) 48.38 68.83 NFX AUC (µg*hr/L) 374.4 347.5 L752F2 - Dose: 7.6 mg - Age 14 days PK Parameters RFX SFX FX AUC (µg*hr/L) 1159.8 1799.3 FX Elimination Half-life (hr) 35.9 56.6 FX Volume of Distribution 34.02 55.6 Steady State (L/kg) FX Clearance (ml/min/kg) 7.17 4.52 FX Mean Residence Time (hr) 41.2 64.5 NFX AUC (µg*hr/L) 333.7 263.5 L752F1 - Dose: 7.9 mg - Age 14 days PK Parameters RFX SFX FX AUC (µg*hr/L) 1030.5 2160.5 FX Elimination Half-life (hr) 25.7 44.1 FX Volume of Distribution 21.5 59.9 Steady State (L/kg) FX Clearance (ml/min/kg) 8.17 3.83 FX Mean Residence Time (hr) 41.5 61.8 NFX AUC (µg*hr/L) 320.5 299.1 L325F1 - Dose: 8.6 mg - Age 15 days PK Parameters RFX SFX FX AUC (µg*hr/L) 1318.48 1891.7 FX Elimination Half-life (hr) 36.2 52.4 FX Volume of Distribution 25.55 44.37 Steady State (L/kg) FX Clearance (ml/min/kg) 6.17 4.33 FX Mean Residence Time (hr) 48.3 68.4 NFX AUC (µg*hr/L) 278.3 283.2 L645F1 - Dose: 7.3 mg - Age 14 days PK Parameters RFX SFX FX AUC (µg*hr/L) 1205.9 1931.1 FX Elimination Half-life (hr) 27.4 44.5 FX Volume of Distribution 27.37 52.17 Steady State (L/kg)  S/R 2.07 1.58  FX 3978.17 42.47  2.44  15.2  0.48 1.42 1.71  4.18 60.743 721.9  S/R 1.55 1.58  FX 3021.3 44.7  1.63  19.31  0.64 1.57 1.26  5.52 58.3 598.3  S/R 2.10 1.72  FX 3142.7 36.8  2.79  15.8  0.48 1.49 1.08  5.29 49.7 619.6  S/R 1.43 1.45  FX 3285.6 41.5  1.74  16.94  0.70 1.42 1.02  5.07 55.6 561.6  S/R 1.60 1.63  FX 3083.3 38.29  1.91  17.59  178  FX Clearance (ml/min/kg) FX Mean Residence Time (hr) NFX AUC (µg*hr/L)  6.83 44.84 405.5  4.17 62.91 400.5  0.62 1.40 1.19  5.33 54.25 806.1  1.3 Lambs in Cohort #3 L740M - Dose: 12.8 mg - Age 32 days PK Parameters RFX SFX FX AUC (µg*hr/L) 1215.8 4171.9 FX Elimination Half-life (hr) 32.9 54.6 FX Volume of Distribution Steady 18.7 38.8 State (L/kg) FX Clearance (ml/min/kg) 6.83 1.99 FX Mean Residence Time (hr) 47.2 78.3 NFX AUC (µg*hr/L) 396.2 557.1 L741M - Dose: 11.8 mg - Age 30 days PK Parameters RFX SFX FX AUC (µg*hr/L) 1815.7 4154.2 FX Elimination Half-life (hr) 35.8 52.9 FX Volume of Distribution Steady 12.1 36.1 State (L/kg) FX Clearance (ml/min/kg) 4.49 2.33 FX Mean Residence Time (hr) 50.4 65.5 NFX AUC (µg*hr/L) 501.3 719.2 L310M - Dose: 13.1 mg - Age 31 days PK Parameters RFX SFX FX AUC (µg*hr/L) 607.1 1942.3 FX Elimination Half-life (hr) 21.4 28.1 FX Volume of Distribution Steady 29.0 38.9 State (L/kg) FX Clearance (ml/min/kg) 13.7 4.17 FX Mean Residence Time (hr) 23.6 56.4 NFX AUC (µg*hr/L) 367.7 484.3 L82M - Dose: 13.3 mg - Age 31 days PK Parameters RFX SFX FX AUC (µg*hr/L) 385.5 2209.9 FX Elimination Half-life (hr) 26.9 54.4 FX Volume of Distribution Steady 34.5 85.6 State (L/kg) FX Clearance (ml/min/kg) 21.5 3.67 FX Mean Residence Time (hr) 33.0 76.3 NFX AUC (µg*hr/L) 240.5 363.9 L310DF - Dose: 13.2 mg - Age 33 days PK Parameters RFX SFX FX AUC (µg*hr/L) 925.2 4981.3 FX Elimination Half-life (hr) 39.3 57.8 FX Volume of Distribution Steady 13.8 36.6  S/R 3.43 1.66  FX 5312.6 47.5  2.07  12.8  0.29 1.66 1.41  3.17 68.2 1053.3  S/R 2.29 1.48  FX 6569.8 54.9  2.97  10.4  0.44 1.30 1.43  2.53 68.5 1320.5  S/R 3.20 1.31  FX 2438.5 31.0  1.34  16.8  0.31 2.39 1.32  6.83 41.1 851.9  S/R 5.73 2.03  FX 2369.1 41.9  2.48  24.5  0.17 2.31 1.51  7.04 58.1 504.4  S/R 5.38 1.47 2.65  FX 5391.1 36.7 9.19 179  State (L/kg) FX Clearance (ml/min/kg) 8.99 1.67 0.19 FX Mean Residence Time (hr) 33.8 68.6 2.03 NFX AUC (µg*hr/L) 395.6 736.2 1.86 L645M1 - Dose: 8.6 mg - Age 33 days PK Parameters RFX SFX S/R FX AUC (µg*hr/L) 762.7 1742.6 2.28 FX Elimination Half-life (hr) 33.3 39.5 1.18 FX Volume of Distribution Steady 32.0 41.4 1.29 State (L/kg) FX Clearance (ml/min/kg) 10.8 4.67 0.44 FX Mean Residence Time (hr) 31.6 55.8 1.77 NFX AUC (µg*hr/L) 413.0 622.9 1.51 L741F - Dose: 12.8 mg - Age 32 Days PK Parameters RFX SFX S/R FX AUC (µg*hr/L) 1236.2 2232.4 1.81 FX Elimination Half-life (hr) 35.4 36.5 1.03 FX Volume of Distribution 20.3 36.7 1.81 Steady State (L/kg) FX Clearance (ml/min/kg) 6.83 3.67 0.55 FX Mean Residence Time (hr) 35.3 55.2 1.56 NFX AUC (µg*hr/L) 342.1 470.3 1.37 L740F - Dose: 8.1 mg - Age 31 days PK Parameters RFX SFX S/R FX AUC (µg*hr/L) 1412.7 2832.6 2.01 FX Elimination Half-life (hr) 29.7 41.2 1.39 FX Volume of Distribution Steady 20.1 41.7 2.08 State (L/kg) FX Clearance (ml/min/kg) 5.83 2.83 0.50 FX Mean Residence Time (hr) 46.8 58.8 1.26 NFX AUC (µg*hr/L) 399.8 565.0 1.41 L82F - Dose: 13.6 mg - Age 38 days PK Parameters RFX SFX S/R FX AUC (µg*hr/L) 841.2 2266.9 2.69 FX Elimination Half-life (hr) 21.2 35.1 1.66 FX Volume of Distribution Steady 19.8 33.5 1.69 State (L/kg) FX Clearance (ml/min/kg) 9.83 3.67 0.37 FX Mean Residence Time (hr) 28.1 44.8 1.59 NFX AUC (µg*hr/L) 373.9 476.2 1.27 PRE03F - Dose: 13.8 mg - Age 36 dats PK Parameters RFX SFX S/R FX AUC (µg*hr/L) 1398.9 1995.2 1.43 FX Elimination Half-life (hr) 24.1 28.7 1.19 FX Volume of Distribution Steady 19.4 67.6 3.48 State (L/kg)  3.18 49.5 1131.8 FX 2362.9 28.6 17.0 7.03 40.2 1336.0 FX 3445.7 35.1 12.9 4.83 44.5 812.4 FX 4240.9 39.9 13.5 4.04 57.1 1064.8 FX 2921.0 25.2 11.6 5.67 33.8 750.0 FX 2390.8 27.8 15.3  180  FX Clearance (ml/min/kg) 20.5 4.05 FX Mean Residence Time (hr) 27.3 39.3 NFX AUC (µg*hr/L) 280.1 453.1 L752F - Dose: 18.6mg - Age 38 days PK Parameters RFX SFX FX AUC (µg*hr/L) 1014.2 1265.3 FX Elimination Half-life (hr) 22.3 34.3 FX Volume of Distribution Steady 19.1 36.3 State (L/kg) FX Clearance (ml/min/kg) 8.17 6.53 FX Mean Residence Time (hr) 24.1 36.8 NFX AUC (µg*hr/L) 339.5 513.3 L645F1 - Dose: 13.5 mg - 38 days PK Parameters RFX SFX FX AUC (µg*hr/L) 1754.4 1994.1 FX Elimination Half-life (hr) 38.6 39.4 FX Volume of Distribution Steady 26.8 27.9 State (L/kg) FX Clearance (ml/min/kg) 4.67 4.13 FX Mean Residence Time (hr) 49.0 53.5 NFX AUC (µg*hr/L) 322.6 501.7  0.20 1.44 1.62  6.84 37.1 721.2  S/R 1.25 1.54  FX 2262.8 19.1  1.90  8.48  0.80 1.52 1.51  7.38 19.1 1052.8  S/R 1.14 1.02  FX 3730.8 38.3  1.04  13.6  0.88 1.09 1.55  4.52 50.7 924.4  1.4 Lambs in Cohort #4 L52M - Dose: 22.1 mg - Age 94 days PK Parameters RFX SFX FX AUC (µg*hr/L) 439.0 723.1 FX Elimination Half-life (hr) 12.5 21.9 FX Volume of Distribution Steady 15.23 20.05 State (L/kg) FX Clearance (ml/min/kg) 18.3 11.5 FX Mean Residence Time (hr) 8.80 11.0 NFX AUC (µg*hr/L) 441.4 811.9 L645M1 - Dose: 21.2 mg - Age 106 days PK Parameters RFX SFX S/R FX AUC (µg*hr/L) 426.1 721.3 1.69 FX Elimination Half-life (hr) 8.46 8.96 1.06 FX Volume of Distribution 15.3 21.38 1.40 Steady State (L/kg) FX Clearance (ml/min/kg) 18.6 11.43 0.59 FX Mean Residence Time (hr) 9.10 11.0 1.21 NFX AUC (µg*hr/L) 457.4 714.8 1.56 L620M - Dose: 19.8 mg - Age 95 days PK Parameters RFX SFX S/R FX AUC (µg*hr/L) 241.7 390.3 1.61 FX Elimination Half-life (hr) 5.67 7.99 1.41 FX Volume of Distribution 25.1 27.9 1.12  S/R 1.65 1.76  FX 1241.1 8.37  1.32  7.21  0.61 1.25 1.84  13.5 8.95 1250.9 FX 1214.0 8.41 7.58 13.7 9.20 1384.2 FX 626.6 6.93 13.4 181  Steady State (L/kg) FX Clearance (ml/min/kg) 35.0 19.9 0.62 FX Mean Residence Time (hr) 6.76 9.79 1.45 NFX AUC (µg*hr/L) 190.6 378.5 1.99 L740M - Dose: 21.5 mg - Age 94 days PK Parameters RFX SFX S/R FX AUC (µg*hr/L) 398.1 664.5 1.67 FX Elimination Half-life (hr) 14.05 24.57 1.75 FX Volume of Distribution 25.92 70.35 2.71 Steady State (L/kg) FX Clearance (ml/min/kg) 19.9 12.6 0.60 FX Mean Residence Time (hr) 17.22 28.00 1.63 NFX AUC (µg*hr/L) 366.6 467.7 1.28 L82M - Dose: 27.3 mg - Age 96 days PK Parameters RFX SFX S/R FX AUC (µg*hr/L) 679.8 1794.6 2.64 FX Elimination Half-life (hr) 25.01 36.46 1.46 FX Volume of Distribution 25.16 61.51 2.44 Steady State (L/kg) FX Clearance (ml/min/kg) 12.2 4.71 0.38 FX Mean Residence Time 31.81 45.15 1.42 (hr) NFX AUC (µg*hr/L) 333.3 625.3 1.88 L741M - Dose: 20.1 mg - Age 96 days PK Parameters RFX SFX S/R FX AUC (µg*hr/L) 362.47 884.98 2.44 FX Elimination Half-life (hr) 12.00 18.99 1.58 FX Volume of Distribution 21.27 55.03 2.59 Steady State (L/kg) FX Clearance (ml/min/kg) 21.7 9.33 0.41 FX Mean Residence Time (hr) 14.95 23.82 1.59 NFX AUC (µg*hr/L) 416.8 604.5 1.45 L82F - Dose: 28.3 mg - Age 99 days PK Parameters RFX SFX FX AUC (µg*hr/L) 536.1 1358.6 FX Elimination Half-life (hr) 15.1 17.7 FX Volume of Distribution Steady 16.9 30.0 State (L/kg) FX Clearance (ml/min/kg) 10.3 6.18 FX Mean Residence Time (hr) 16.1 23.0 NFX AUC (µg*hr/L) 392.5 655.4 L740F - Dose: 16 mg - Age 99 days PK Parameters RFX SFX S/R FX AUC (µg*hr/L) 419.6 778.7 1.86 FX Elimination Half-life (hr) 12.0 16.2 1.35 FX Volume of Distribution Steady 19.1 37.3 1.95  26.5 8.37 568.4 FX 954.5 14.31 18.23 17.4 17.40 1021.6 FX 2494.2 36.36 18.15 6.75 45.27 1156.2 FX 1357.20 13.76 12.01 12.4 16.30 1004.5 S/R 2.53 1.17  FX 1937.3 17.0  1.77  10.6  0.39 1.43 1.67  8.77 20.6 1245.3 FX 1279.0 11.3 10.9 182  State (L/kg) FX Clearance (ml/min/kg) 18.4 10.8 FX Mean Residence Time (hr) 11.7 15.9 NFX AUC (µg*hr/L) 300.4 528.4 PRE03F - Dose: 30.6 mg - Age 91 days PK Parameters RFX SFX FX AUC (µg*hr/L) 498.17 723.53 FX Elimination Half-life (hr) 26.85 39.06 FX Volume of Distribution 29.72 74.11 Steady State (L/kg) FX Clearance (ml/min/kg) 16.7 11.5 FX Mean Residence Time (hr) 21.50 36.92 NFX AUC (µg*hr/L) 548.0 728.4 L77F1 - Dose: 27 mg - Age 111 days PK Parameters RFX SFX FX AUC (µg*hr/L) 428.1 1053.6 FX Elimination Half-life (hr) 13.0 22.1 FX Volume of Distribution 17.5 39.0 Steady State (L/kg) FX Clearance (ml/min/kg) 18.4 7.83 FX Mean Residence Time (hr) 13.7 20.5 NFX AUC (µg*hr/L) 483.3 638.7 L752F - Dose: 19.8 mg - Age 91 days PK Parameters RFX SFX FX AUC (µg*hr/L) 269.0 454.5 FX Elimination Half-life (hr) 6.49 7.51 FX Volume of Distribution Steady 21.2 28.1 State (L/kg) FX Clearance (ml/min/kg) 30.6 18.5 FX Mean Residence Time (hr) 7.58 9.67 NFX AUC (µg*hr/L) 394.8 701.4 L325F1 - Dose: 17 mg - Age 110 days PK Parameters RFX SFX FX AUC (µg*hr/L) 354.6 424.1 FX Elimination Half-life (hr) 9.87 17.3 FX Volume of Distribution Steady 13.7 21.6 State (L/kg) FX Clearance (ml/min/kg) 23.3 18.3 FX Mean Residence Time (hr) 6.35 10.33 NFX AUC (µg*hr/L) 214.7 392.8 1.5 Lambs in Cohort #5 L740M - Dose: 37.7 mg - Age 220 days Pk Parameters RFX SFX FX AUC (µg*hr/L) 381.6 679.0  0.54 1.36 1.76  13.4 14.0 1162.2  S/R 1.45 1.45  FX 1202.4 29.56  2.49  21.32  0.69 1.72 1.33  13.8 25.64 1376.2  S/R 2.46 1.70  FX 1498.6 14.1  2.22  12.2  0.41 1.50 1.32  11.3 18.4 1099.2  S/R 1.69 1.16  FX 701.4 6.75  1.32  12.0  0.59 1.28 1.78  23.8 8.44 1096.2  S/R 1.20 1.76  FX 923.9 7.12  1.57  6.75  0.84 1.62 1.83  18.4 6.23 601.8  S/R 1.78  FX 1064.3  183  FX Elimination Half-life (hr) 16.4 24.9 1.52 FX Volume of Distribution 30.1 63.4 2.10 Steady State (L/kg) FX Clearance (ml/min/kg) 21.7 12.2 0.56 FXMean Residence Time (hr) 20.4 24.1 1.18 NFX AUC (µg*hr/L) 539.5 743.0 1.37 L82M - Dose: 39.9 mg - Age 219 days Pk Parameters RFX SFX S/R FX AUC (µg*hr/L) 338.2 1259.3 3.72 FX Elimination Half-life (hr) 8.01 14.9 1.87 FX Volume of Distribution 9.20 12.1 1.32 Steady State (L/kg) FX Clearance (ml/min/kg) 23.3 6.53 0.27 FXMean Residence Time (hr) 6.10 13.5 2.23 NFX AUC (µg*hr/L) 628.7 828.2 1.32 L741M - Dose: 32.4 mg - Age 225 days Pk Parameters RFX SFX S/R FX AUC (µg*hr/L) 547.0 1387.7 2.53 FX Elimination Half-life (hr) 8.42 13.3 1.58 FX Volume of Distribution 12.6 16.1 1.28 Steady State (L/kg) FX Clearance (ml/min/kg) 15.3 6.04 0.39 FXMean Residence Time (hr) 8.84 17.5 1.98 NFX AUC (µg*hr/L) 491.8 746.6 1.51 L310OPM - Dose: 48.2 mg - Age 246 days Pk Parameters RFX SFX S/R FX AUC (µg*hr/L) 525.5 1364.0 2.59 FX Elimination Half-life (hr) 10.1 14.8 1.46 FX Volume of Distribution Steady 8.95 32.0 3.57 State (L/kg) FX Clearance (ml/min/kg) 15.8 6.19 0.38 FXMean Residence Time (hr) 12.2 16.8 1.37 NFX AUC (µg*hr/L) 775.5 1216.6 1.56 L82F - Dose: 38.4 mg - Age 182 days Pk Parameters RFX SFX S/R FX AUC (µg*hr/L) 480.1 1568.4 3.26 FX Elimination Half-life (hr) 15.5 24.6 1.5 FX Volume of Distribution Steady 13.3 35.1 2.63 State (L/kg) FX Clearance (ml/min/kg) 16.7 5.19 0.31 FXMean Residence Time (hr) 16.8 20.9 1.24 NFX AUC (µg*hr/L) 656.0 932.0 1.42 L740F - Dose: 22mg - Age 212 days Pk Parameters RFX SFX S/R FX AUC (µg*hr/L) 575.5 914.4 1.58 FX Elimination Half-life (hr) 8.00 10.3 1.29  25.8 20.7 15.7 22.0 1071.5 FX 2367.5 10.5 3.00 7.06 7.11 1513.8 FX 1881.7 12.6 8.15 8.87 15.3 1272.3 FX 2093.5 10.6 5.46 7.99 11.4 1986.3 FX 1911.1 16.4 11.1 8.72 21.2 1773.2 FX 1434.5 8.16 184  FX Volume of Distribution 8.89 13.9 Steady State (L/kg) FX Clearance (ml/min/kg) 14.6 9.04 FXMean Residence Time (hr) 7.02 14.1 NFX AUC (µg*hr/L) 551.7 921.1 PRE03F - Dose: 43.5 mg - Age 199 days Pk Parameters RFX SFX FX AUC (µg*hr/L) 435.4 880.4 FX Elimination Half-life (hr) 10.1 19.0 FX Volume of Distribution 14.9 32.9 Steady State (L/kg) FX Clearance (ml/min/kg) 18.4 9.43 FXMean Residence Time (hr) 13.1 22.9 NFX AUC (µg*hr/L) 506.9 802.5 L91F - Dose: 44.2 mg - Age 167 days Pk Parameters RFX SFX FX AUC (µg*hr/L) 474.7 720.6 FX Elimination Half-life (hr) 23.7 24.8 FX Volume of Distribution 33.9 61.1 Steady State (L/kg) FX Clearance (ml/min/kg) 18.7 11.6 FXMean Residence Time (hr) 24.4 29.0 NFX AUC (µg*hr/L) 462.3 887.9  1.56  5.29  0.62 2.01 1.66  11.8 7.58 1650.5  S/R 2.02 1.88  FX 1251.8 11.0  2.20  11.19  0.49 1.74 1.58  13.5 14.0 1563.4  S/R 1.51 1.04  FX 1198.2 24.3  1.80  22.1  0.63 1.18 1.92  13.9 26.4 1208.1  1.6 Lambs in Cohort #6 L740M - Dose: 57.8 mg - Age 417 days PK Parameter RFX SFX S/R FX AUC (µg*hr/L) 419.2 2024.1 4.83 FX Elimination Half-life (hr) 5.53 6.88 1.24 FX Volume of Distribution Steady 1.90 12.74 6.69 State (L/kg) FX Clearance (ml/min/kg) 14.5 4.05 0.28 FX Mean Residence Time (hr) 3.85 5.34 1.39 NFX AUC (µg*hr/L) 681.1 916.3 1.35 L82M - Dose: 62 mg - Age 364 days PK Parameter RFX SFX S/R FX AUC (µg*hr/L) 1478.8 2230.3 1.51 FX Elimination Half-life (hr) 7.49 12.8 1.72 FX Volume of Distribution Steady 1.06 2.88 2.70 State (L/kg) FX Clearance (ml/min/kg) 10.8 3.79 0.34 FX Mean Residence Time (hr) 4.25 17.9 4.22 NFX AUC (µg*hr/L) 1121.5 1406.0 1.25 L741M - Dose: 53.1 mg Age 423 days PK Parameter RFX SFX S/R FX AUC (µg*hr/L) 376.0 1344.4 3.58  FX 2465.0 6.54 1.66 6.86 4.09 1455.3 FX 2364.0 7.81 4.51 4.04 10.6 2811.8 FX 1653.6 185  FX Elimination Half-life (hr) 4.02 8.00 1.99 FX Volume of Distribution Steady 8.03 12.4 1.55 State (L/kg) FX Clearance (ml/min/kg) 17.8 6.18 0.28 FX Mean Residence Time (hr) 4.67 10.7 2.31 NFX AUC (µg*hr/L) 662.2 826.6 1.25 L310OP - Dose: 65.4 mg - Age 365 days PK Parameter RFX SFX S/R FX AUC (µg*hr/L) 427.0 1185.6 2.78 FX Elimination Half-life (hr) 12.8 14.0 1.10 FX Volume of Distribution Steady 13.6 35.8 2.63 State (L/kg) FX Clearance (ml/min/kg) 13.2 7.13 0.36 FX Mean Residence Time (hr) 9.29 16.1 1.74 NFX AUC (µg*hr/L) 758.8 1089.4 1.44 L825M2 - Dose: 65.1 mg - Age 389 days PK Parameter RFX SFX S/R FX AUC (µg*hr/L) 343.8 668.8 1.95 FX Elimination Half-life (hr) 6.40 10.5 1.65 FX Volume of Distribution Steady 17.9 20.7 1.16 State (L/kg) FX Clearance (ml/min/kg) 17.5 9.95 0.52 FX Mean Residence Time (hr) 7.14 12.0 1.68 NFX AUC (µg*hr/L) 197.9 494.8 2.50 L82F - Dose: 60.6 mg - Age 385 days PK Parameter RFX SFX S/R FX AUC (µg*hr/L) 908.1 1305.8 1.44 FX Elimination Half-life (hr) 9.98 12.4 1.25 FX Volume of Distribution Steady 3.22 11.0 3.42 State (L/kg) FX Clearance (ml/min/kg) 10.2 6.35 0.62 FX Mean Residence Time (hr) 4.21 14.36 3.41 NFX AUC (µg*hr/L) 726.7 1151.3 1.58 L740F - Dose: 44.9 mg - Age 411 days PK Parameter RFX SFX S/R FX AUC (µg*hr/L) 392.7 833.3 2.12 FX Elimination Half-life (hr) 4.48 6.19 1.38 FX Volume of Distribution Steady 9.66 16.78 1.74 State (L/kg) FX Clearance (ml/min/kg) 18.5 10.3 0.56 FX Mean Residence Time (hr) 6.59 8.05 1.22 NFX AUC (µg*hr/L) 726.6 965.1 1.33 L741F - Dose: 50.8 mg - Age 436 days PK Parameter RFX SFX S/R FX AUC (µg*hr/L) 406.7 1136.0 2.79  6.45 5.09 9.2 8.41 1502.5 FX 1537.1 10.6 8.77 9.8 13.4 1973.0 FX 994.6 8.46 9.79 12.8 9.74 691.8 FX 2294.1 10.0 4.42 7.35 10.14 1798.0 FX 1227.2 5.84 6.02 12.7 7.39 1856.1 FX 1363.9  186  FX Elimination Half-life (hr) 6.80 8.50 1.25 FX Volume of Distribution Steady 6.50 20.06 3.08 State (L/kg) FX Clearance (ml/min/kg) 17.3 7.43 0.36 FX Mean Residence Time (hr) 7.39 9.16 1.24 NFX AUC (µg*hr/L) 770.6 972.45 1.26 L752F - Dose: 51.9 mg - Age 386 days PK Parameter RFX SFX S/R FX AUC (µg*hr/L) 689.5 850.07 1.23 FX Elimination Half-life (hr) 6.82 8.99 1.32 FX Volume of Distribution Steady 6.89 9.10 1.32 State (L/kg) FX Clearance (ml/min/kg) 10.4 6.32 0.80 FX Mean Residence Time (hr) 6.10 9.74 1.60 NFX AUC (µg*hr/L) 185.7 504.2 2.71  6.96 6.27 9.3 8.56 1732.5 FX 1800.5 9.43 3.41 7.35 6.15 689.9  187  Appendix	
  2	
  Urine	
  data	
  for	
  individual	
  lambs	
   2.1 Lambs in Cohort #1 L82M Cl Xu 5.9kg (ml/min/kg) SFX 56.3 0.132 RFX 39.4 0.0395 TFMP 33.4 SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L310DF Cl Xu 4.1kg (ml/min/kg) SFX 90.9 0.118 RFX 55.2 00.154 TFMP 34.4 SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L740M Cl Xu 4.9kg (ml/min/kg) SFX 83.2 0.116 RFX 39.4 0.198 TFMP 30.5 SNFX RNFX SFXGluc RFXGluc SNFX-  %dose 1.43 0.832 0.889 %dose 1.23 0.747 0.931 %dose 1.69 0.799 1.23 -  L740F 3.3kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L741F 5.0kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L77F1 6.1kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFX-  Xu  Cl (ml/min/kg)  %dose  84.0 60.4 40.4 -  0.265 0.305 -  2.58 0.936 2.49 -  -  -  -  -  -  -  -  -  -  -  -  -  Xu  Cl (ml/min/kg)  %dose  92.3 54.2 49.2 -  0.213 0.172 -  2.10 1.23 2.12 -  -  -  -  -  -  -  -  -  -  -  -  -  104.8 59.7 46.5 -  Cl (ml/min/kg) 0.193 0.349 -  -  -  -  -  -  -  -  -  -  Xu  %dose 1.72 0.979 1.52 -  188  Gluc RNFXGluc L620M 5.5kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L620M 5.5kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc  -  -  -  Xu  Cl (L/hr/kg)  %dose  64.8 46.4 36.2 -  0.149 0.137 -  2.49 0.769 1.73 -  -  -  -  -  -  -  -  -  -  -  -  -  59.7 48.4 39.7 -  Cl (ml/min/kg) 0.0673 0.154 -  -  -  -  -  -  -  -  -  -  -  -  -  Xu  2.2 Lambs in Cohort #2 L82M Cl Xu 7.3 kg (ml/min/kg) SFX 56.4 0.133 RFX 39.4 0.0394 TFMP 66.8 SNFX RNFX -  Gluc RNFXGluc L752F1 5.0kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc  -  -  -  96.7 54.7 48.2 -  Cl (L/hr/kg) 0.283 0.354 -  -  -  -  -  -  -  -  -  -  -  -  -  Xu  %dose 2.42 1.18 2.41 -  %dose 1.09 0.881 1.44 -  %dose 1.24 0.734 1.24 -  L740F 5.1 kg SFX RFX TFMP SNFX RNFX  Xu 95.6 54.9 73.1 -  Cl (ml/min/kg) 0.116 0.138 -  %dose 1.88 0.00419 1.43 -  189  SFXGluc RFXGluc SNFXGluc RNFXGluc L310DF 7.4 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L741M 7.1 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L740M 7.4 kg SFX RFX  -  -  -  -  -  -  -  -  -  -  -  -  90.9 55.2 68.9 -  Cl (ml/min/kg) 0.118 0.151 -  -  -  -  -  -  -  -  -  -  -  -  -  Xu  %dose 1.23 0.747 0.931 -  88.6 48.5 89.1 -  Cl (ml/min/kg) 0.0573 0.114 -  -  -  -  -  -  -  -  -  -  -  -  -  Xu  Xu 88.2 65.4  Cl (ml/min/kg) 0.764 0.149  %dose 1.24 0.683 1.25 -  %dose 1.24 0.922  SFXGluc RFXGluc SNFXGluc RNFXGluc L77F1 7.9 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L752F1 12.8 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L752F2 7.6 kg SFX RFX  -  -  -  -  -  -  -  -  -  -  -  -  104 59.7 92.9 -  Cl (ml/min/kg) 0.193 0.349 -  -  -  -  -  -  -  -  -  -  -  -  -  Xu  %dose 1.72 0.979 1.52 -  93.9 67.8 104 -  Cl (ml/min/kg) 0.0917 1.14 -  -  -  -  -  -  -  -  -  -  -  -  -  Xu  Xu 92.9 65.4  Cl (ml/min/kg) 0.119 0.129  %dose 1.19 0.859 1.27 -  %dose 1.27 0.896 190  TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L620M 7.8 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L645M 8.6 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc  80.9 -  -  1.14 -  -  -  -  -  -  -  -  -  -  -  -  -  81.4 53.5 63.4 -  Cl (ml/min/kg) 0.0575 0.0697 -  -  -  -  -  -  -  -  -  -  -  -  -  Xu  108 -  -  1.43 -  -  -  -  -  -  -  -  -  -  -  -  -  %dose 1.12 0.734 0.863 -  86.8 65.6 71.7 -  Cl (ml/min/kg) 0.0648 0.0889 -  -  -  -  -  -  -  -  -  -  -  -  -  Xu  TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc  %dose 1.11 0.841 0.919 -  191  2.3 Lambs in Cohort #3 L740M Cl L740F Xu %dose 12.8 kg (ml/min/kg) 8.1 kg SFX 121 0.0135 0.562 SFX RFX 76.0 0.157 0.353 RFX TFMP 154 0.717 TFMP SNFX 205 0.958 SNFX RNFX 126 0.588 RNFX SFXSFX101 0.473 Gluc Gluc RFXRFX125 0.397 Gluc Gluc SNFXSNFX138 0.646 Gluc Gluc RNFXRNFX177 0.360 Gluc Gluc L741M 11.8 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L82M 13.3 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc  Xu 115 84.3 119 182 126 91.1 89.7 127 135 Xu 118 70.2 112 186 115. 970 100. 729 114  Cl L82F %dose (ml/min/kg) 13.6 kg 0.105 0.574 SFX 0.206 0.420 RFX 0.592 TFMP 0.908 SNFX 0.630 RNFX SFX0.454 Gluc RFX0.399 Gluc SNFX0.636 Gluc RNFX0.433 Gluc Cl L77F1 %dose (ml/min/kg) 12.8 kg 0.0368 0.434 SFX 0.0723 0.257 RFX 0.413 TFMP 0.683 SNFX -  0.425  -  0.369  -  0.272  RNFX SFXGluc RFXGluc  117 77.1 128 194 106  Cl (ml/min/kg) 0.156 0.217 -  114  -  0.715  166  -  0.414  109  -  0.999  133  -  0.523  Xu  %dose 0.735 0.486 0.805 1.22 0.666  130 96.2 140 176 109  Cl (ml/min/kg) 0.0645 0.113 -  122  -  0.431  195  -  0.339  124  -  0.474  127  -  0.309  Xu  %dose 0.461 0.340 0.498 0.625 0.356  122 88.6 156 125  Cl (ml/min/kg) 0.0729 0.148 -  82.3  -  0.305  125  -  0.465  94.7  -  0.314  Xu  %dose 0.454 0.328 0.578 0.464  192  SNFXGluc RNFXGluc L645M 8.6 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L310M 13.2 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L310DF 13.2 kg SFX RFX TFMP SNFX RNFX SFX-  129  -  0.475  94.3  -  0.309  Xu 131 77.3 131 117 92.4 114 127 138 146 Xu 133 110 141 127 108 101 96.1 121 192 Xu 120 98.3 122 153 123 111  SNFXGluc RNFXGluc  Cl L752F2 %dose (ml/min/kg) 18.6 kg 0.155 0.617 SFX 0.165 0.363 RFX 0.618 TFMP 0.550 SNFX 0.434 RNFX SFX0.539 Gluc RFX0.457 Gluc SNFX0.650 Gluc RNFX0.548 Gluc Cl L325F1 %dose (ml/min/kg) 18.6 kg 0.142 0.603 SFX 0.198 0.500 RFX 0.638 TFMP 0.578 SNFX 0.490 RNFX SFX0.460 Gluc RFX0.390 Gluc SNFX0.552 Gluc RNFX0.422 Gluc  143  -  0.531  112  -  0.414  Xu 137 106 122 172 144  Cl (ml/min/kg) 0.253 0.336 -  %dose 0.697 0.537 0.619 0.869 0.728  136  -  0.691  153  -  0.470  147  -  0.743  176  -  0.639  130 108 134 130 105  Cl (ml/min/kg) 0.328 0.275 -  102  -  0.604  82.3  -  0.484  118  -  0.697  158  -  0.522  Xu  %dose 0.765 0.638 0.792 0.768 0.622  Cl %dose (ml/min/kg) 0.278 0.606 0.362 0.497 0.632 0.774 0.625 0.564 193  Gluc RFXGluc SNFXGluc RNFXGluc  121  -  0.311  120  -  0.609  105  -  0.531  2.4 Lambs in Cohort #4 L620M Cl Xu 19.8 kg (ml/min/kg) SFX 82.5 0.0538 RFX 53.7 0.126 TFMP 118 SNFX 23.5 RNFX 15.0 SFX9.14 Gluc RFX7.57 Gluc SNFX14.1 Gluc RNFX19.0 Gluc L741M 20.1 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L740M 21.5 kg SFX RFX  %dose 0.630 0.410 0.905 0.180 0.011 0.070 0.058 0.108 0.069  95.3 53.8 94.6 20.7 12.9  Cl (ml/min/kg) 0.0354 0.0575 -  11.6  -  0.099  7.63  -  0.065  15.1  -  0.129  29.4  -  0.080  Xu  Xu 109 68.5  Cl (ml/min/kg) 0.0354 0.0677  %dose 0.808 0.457 0.802 0.176 0.110  %dose 0.857 0.536  L740F 16 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L771F 27 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc PRE03F1 30.6 kg SFX RFX  90.1 79.0 106 18.5 8.40  Cl (ml/min/kg) 0.546 0.239 -  14.6  -  0.182  19.0  -  0.111  20.4  -  0.254  13.0  -  0.163  Xu  %dose 1.11 0.989 1.31 0.230 0.101  87.2 57.8 96.1 23.9 16.5  Cl (ml/min/kg) 0.0554 0.0738 -  13.7  -  0.107  17.4  -  0.058  32.2  -  0.252  46.1  -  0.126  Xu  Xu 89.6 61.3  Cl (ml/min/kg) 0.0553 0.0572  %dose 0.681 0.452 0.751 0.188 0.129  %dose 0.685 0.445 194  TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L82M 27.3 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L645M1 21.2 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc  109 17.2 8.15  -  0.857 0.135 0.064  12.9  -  0.102  16.8  -  0.054  15.6  -  0.122  17.3  -  0.058  93.6 69.9 116 24.6 14.1  Cl (ml/min/kg) 0.0206 0.107 -  17.2  -  0.131  20.4  -  0.079  35.2  -  0.267  46.6  -  0.126  Xu  %dose 0.709 0.529 0.884 0.187 0.108  96.8 73.4 127 38.8 16.0  Cl (ml/min/kg) 0.0703 0.121 -  11.6  -  0.084  18.8  -  0.064  25.9  -  0.188  20.5  -  0.076  Xu  TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L82F 28.3 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc  99.7 25.0 14.9  -  0.723 0.181 0.108  16.6  -  0.120  18.8  -  0.064  32.3  -  0.234  43.9  -  0.101  94.8 58.0 112 12.9 7.06  Cl (ml/min/kg) 0.0538 0.0903 -  17.7  -  0.131  14.9  -  0.110  31.7  -  0.233  42.5  -  0.166  Xu  %dose 0.697 0.427 0.826 0.095 0.052  %dose 0.702 0.540 0.924 0.282 0.116  195  2.5 Lambs Cohort #5 L310OPM Cl Xu 48.2 kg (ml/min/kg) SFX 134 0.295 RFX 94.8 0.0632 TFMP 127 SNFX 214 RNFX 142 SFX-Gluc 113 RFX-Gluc 175 SNFX146 Gluc RNFX178 Gluc L740M 37.7 kg SFX RFX TFMP SNFX RNFX SFX-Gluc RFX-Gluc SNFXGluc RNFXGluc  %dose 0.238 0.196 0.264 0.445 0.295 0.236 0.156 0.304 0.204  231 181 244 286 241 174 222  Cl (ml/min/kg) 0.153 0.214 -  209  -  0.555  185  -  0.493  Xu  %dose 0.613 0.480 0.649 0.760 0.639 0.463 0.323  L741M 32.4 kg SFX RFX TFMP SNFX RNFX SFX-Gluc  125 79.4 137 214 131. 154  Cl (ml/min/kg) 0.04658 0.0748 -  RFX-Gluc  202  -  175  -  0.542  222  -  0.376  SNFXGluc RNFXGluc  Xu  %dose 0.385 0.245 0.424 0.662 0.404 0.481 0.317 248  L91F 44.2 kg SFX RFX TFMP SNFX RNFX SFX-Gluc RFX-Gluc SNFXGluc RNFXGluc L740F 22.0 kg SFX RFX TFMP SNFX RNFX SFX-Gluc RFX-Gluc SNFXGluc RNFXGluc  147 85.7 128 215 110 123 173  Cl (ml/min/kg) 0.0779 0.0689 -  123  -  0.279  182  -  0.186  Xu  %dose 0.334 0.193 0.296 0.487 0.249 0.279 0.166  119 95.8 114 210 167 229 276  Cl (ml/min/kg) 0.0995 0.127 -  101  -  0.461  186  -  0.395  Xu  %dose 0.546 0.436 0.519 0.956 0.760 0.589 0.349  L82F 38.4 kg SFX RFX TFMP SNFX RNFX SFX-Gluc  134 92.9 120 217 154 243  Cl (ml/min/kg) 0.0385 0.0845 -  RFX-Gluc  204  -  0.273  246  -  0.642  264  -  0.428  SNFXGluc RNFXGluc  Xu  %dose 0.351 0.242 0.312 0.567 0.402 0.373  196  L82M 39.9 kg SFX RFX TFMP SNFX RNFX SFX-Gluc RFX-Gluc SNFXGluc RNFXGluc  153 97.6 186 188 119 181 226  Cl (ml/min/kg) 0.0517 0.121 -  217  -  0.545  269  -  0.423  Xu  2.6 Lambs in Cohort #6 L310OPM Cl Xu 65.4 kg (ml/min/kg) SFX 176 0.0389 RFX 108 0.0515 TFMP 165 SNFX 235 RNFX 157 -  %dose 0.385 0.244 0.466 0.473 0.298 0.455 0.316  %dose 0.269 0.165 0.252 0.359 0.241  SFX-Gluc  164  -  0.250  RFX-Gluc  156  -  0.242  SNFX-Gluc  163  -  0.249  RNFXGluc  172  -  0.256  L310NonO pM 60.7 kg SFX RFX TFMP SNFX RNFX  200 127 161 220 125  Cl (ml/min/kg) 0.0387 0.0818 -  SFX-Gluc  196  -  0.323  RFX-Gluc  198  -  0.361  SNFX-Gluc  171  -  0.282  RNFX-  199  -  0.363  Xu  %dose 0.329 0.209 0.265 0.362 0.205  PRE03F1 43.5 kg SFX RFX TFMP SNFX RNFX SFX-Gluc RFX-Gluc SNFXGluc RNFXGluc L741F 50.8 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L82F 60.6 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFX-  124 83.3 145 169 126 111 174  Cl (ml/min/kg) 0.0548 0.0732 -  209  -  0.482  142  -  0.326  Xu  %dose 0.286 0.192 0.335 0.391 0.291 0.255 0.172  183 144 189 211 165  Cl (ml/min/kg) 0.0523 0.0887 -  130  -  0.256  142  -  0.341  171  -  0.336  161  -  0.268  Xu  %dose 0.361 0.283 0.372 0.415 0.324  195 141 215 252 157  Cl (ml/min/kg) 0.0353 0.0886 -  164  -  0.270  171  -  0.299  140  -  0.230  155  -  0.257  Xu  %dose 0.322 0.233 0.355 0.416 0.258  197  Gluc  Gluc  L740M 57.8 kg SFX RFX TFMP SNFX RNFX  218 132 174 201 127  Cl (ml/min/kg) 0.102 0.116 -  SFX-Gluc  222  -  0.204  RFX-Gluc  250  -  0.260  SNFX-Gluc  169  -  0.243  RNFXGluc  177  -  0.286  Xu  %dose 0.378 0.229 0.301 0.348 0.221  L741M 53.1 kg SFX RFX TFMP SNFX RNFX  168 112 161 165 115  Cl (ml/min/kg) 0.0354 0.0865 -  SFX-Gluc  106  -  0.200  RFX-Gluc  110  -  0.233  SNFX-Gluc  142  -  0.267  RNFXGluc  152  -  0.275  L82M 62 kg SFX RFX TFMP SNFX RNFX SFX-Gluc RFX-Gluc SNFX-Gluc  Xu  Xu 176 144 158 167 122 157 174 189  Cl (ml/min/kg) 0.0352 0.133 -  %dose 0.317 0.211 0.302 0.311 0.217  L742F 51.9 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc L740F 44.9 kg SFX RFX TFMP SNFX RNFX SFXGluc RFXGluc SNFXGluc RNFXGluc  182 118 182 115 77  Cl (ml/min/kg) 0.132 0.169 -  204  -  0.401  198  -  0.381  153  -  0.203  163  -  0.230  Xu  %dose 0.351 0.227 0.351 0.222 0.148  165 107 165 98.7 76.6  Cl (ml/min/kg) 0.0685 0.0864 -  143  -  0.201  164  -  2.88  123  -  0.174  115  -  0.185  Xu  %dose 0.369 0.239 0.342 0.189 0.433  %dose 0.283 0.233 0.255 0.269 0.197 0.294 0.254 0.331  198  RNFXGluc  205  -  0.311  199  Appendix	
  3:	
  Cohorts	
  1	
  to	
  6	
  blood	
  pH	
  and	
  body	
  temperature	
  data.	
   3.1: Cohorts 1 to 6 control experiment blood pH CTL  Cohort #1 Male  Time -15 5 15 30 60 120  AVG 7.39 7.37 7.37 7.38 7.38 7.38  CTL  Cohort #4 Male  Time -15 5 15 30 60 120  AVG 7.49 7.47 7.51 7.48 7.48 7.48  ±SD 0.0441 0.0220 0.0215 0.0354 0.0388 0.0340  ±SD 0.0288 0.0368 0.0712 0.0350 0.0542 0.0376  Cohort #1 Female AVG ±SD 7.48 0.0316 7.45 0.0329 7.45 0.0284 7.44 0.0282 7.44 0.0364 7.44 0.0333  Cohort #2 Male AVG ±SD 7.42 0.0176 7.41 0.0226 7.42 0.0247 7.42 0.0159 7.41 0.0230 7.41 0.0309  Cohort #2 Female AVG ±SD 7.41 0.0294 7.41 0.0297 7.41 0.0354 7.41 0.0414 7.41 0.0363 7.42 0.0161  Cohort #4 Female AVG ±SD 7.45 0.0189 7.45 0.0265 7.44 0.0276 7.44 0.0195 7.46 0.0227 7.46 0.0241  Cohort #5 Male AVG ±SD 7.47 0.0966 7.49 0.0602 7.48 0.0281 7.48 0.0293 7.47 0.0535 7.49 0.0537  Cohort #5 Female AVG ±SD 7.48 0.0555 7.47 0.0462 7.45 0.0217 7.46 0.0565 7.48 0.0485 7.45 0.0281  ±SD 0.0065 0.0218 0.0130 0.0245 0.0081 0.0441  Cohort #3 Female AVG ±SD 7.45 0.0189 7.45 0.0265 7.44 0.0276 7.44 0.0195 7.46 0.0227 7.46 0.0241  Cohort #6 Male AVG ±SD 7.50 0.0360 7.49 0.0255 7.50 0.0330 7.50 0.0371 7.47 0.0248 7.49 0.0318  Cohort #6 Female AVG ±SD 7.48 0.0143 7.49 0.0159 7.49 0.0223 7.47 0.0384 7.48 0.0184 7.50 0.0237  Cohort #3 Male AVG ±SD 7.44 0.0381 7.45 0.0264 7.43 0.0482 7.42 0.0214 7.44 0.0338 7.45 0.0361  Cohort #3 Female AVG ±SD 7.44 0.0303 7.43 0.0208 7.44 0.0280 7.43 0.0181 7.43 0.0484 7.46 0.0268  Cohort #3 Male AVG 7.43 7.42 7.43 7.43 7.44 7.47  3.2 : FX experiment blood pH Time -15 5 15 30 60 120  Cohort #1 Male AVG ±SD 7.36 0.0104 7.33 0.0324 7.36 0.0325 7.36 0.0275 7.35 0.0478 7.35 0.0199 Cohort #4 Male  Time -15 5 15 30 60 120  AVG 7.47 7.49 7.46 7.49 7.46 7.45  ±SD 0.0332 0.0522 0.0494 0.0395 0.0702 0.0629  Cohort #1 Female AVG ±SD 7.41 0.0234 7.40 0.0142 7.41 0.0146 7.40 0.0142 7.40 0.0128 7.40 0.0245 Cohort #4 Female AVG ±SD 7.52 0.0539 7.50 0.0258 7.49 0.0295 7.48 0.0230 7.48 0.0336 7.48 0.0246  Cohort #2 Male AVG ±SD 7.44 0.0218 7.42 0.0255 7.42 0.0220 7.43 0.0296 7.41 0.0137 7.42 0.0183 Cohort #5 Male AVG 7.49 7.48 7.47 7.48 7.47 7.49  ±SD 0.0556 0.0328 0.0165 0.0378 0.0437 0.0634  Cohort #2 Female AVG ±SD 7.43 0.0379 7.43 0.0472 7.43 0.0331 7.42 0.0307 7.41 0.0272 7.41 0.0255 Cohort #5 Female AVG ±SD 7.51 0.0516 7.52 0.0253 7.49 0.0253 7.49 0.0396 7.48 0.0164 7.47 0.0251  Cohort #6 Male AVG 7.50 7.50 7.50 7.50 7.50 7.48  ±SD 0.0298 0.0444 0.0301 0.0255 0.0308 0.0651  Cohort #6 Female AVG ±SD 7.45 0.0410 7.50 0.0278 7.51 0.0463 7.51 0.0530 7.48 0.0130 7.50 0.0500  200  3.3: Cohorts 1 to 6 control experiment temperature Time -15 5 15 30 60 120  AVG 39.475 39.375 39.425 39.500 39.400 39.400  ±SD 0.287 0.419 0.419 0.271 0.432 0.432  Cohort #1 Female AVG ±SD 39.220 0.277 39.500 0.447 39.500 0.464 39.500 0.515 39.460 0.503 39.580 0.487  Time -15 5 15 30 60 120  Cohort #4 Male AVG ±SD 39.700 0.732 39.767 0.671 39.683 0.700 39.700 0.690 39.683 0.714 39.833 0.582  Cohort #4 Female AVG ±SD 39.600 0.356 39.586 0.358 39.700 0.379 39.686 0.460 39.900 0.673 40.000 0.764  Cohort #1 Male  ±SD 0.302 0.396 0.383 0.619 0.402 0.356  Cohort #2 Female AVG ±SD 39.843 0.645 39.614 0.604 39.671 0.512 39.860 0.611 39.740 0.428 39.720 0.466  AVG 39.650 39.650 39.675 39.625 39.600 39.167  ±SD 0.351 0.465 0.465 0.550 0.583 0.058  Cohort #3 Female AVG ±SD 39.683 0.133 39.400 0.219 39.433 0.273 39.567 0.242 39.500 0.261 39.617 0.313  Cohort #5 Male AVG ±SD 39.433 0.208 39.467 0.321 39.500 0.361 39.367 0.252 39.367 0.153 39.300 0.200  Cohort #5 Female AVG ±SD 39.440 0.365 39.500 0.381 39.440 0.439 39.440 0.336 39.420 0.402 39.480 0.370  Cohort #6 Male AVG ±SD 39.250 0.513 39.533 0.350 39.467 0.568 39.683 0.677 39.500 0.901 39.333 0.418  Cohort #6 Female AVG ±SD 38.925 0.492 38.850 0.500 38.800 0.572 38.900 0.497 39.000 0.392 39.100 0.392  Cohort #2 Male AVG 39.750 39.680 39.667 39.833 39.717 39.667  Cohort #3 Male  3.4: Cohorts 1 to 6 FX experiment blood temperature Time -15 5 15 30 60 120  Cohort #1 Male AVG ±SD 39.9 0.591 39.6 0.675 39.3 0.984 39.4 0.779 39.3 0.976 39.5 0.608  Cohort #1 Female AVG ±SD 39.7 0.391 39.6 0.356 39.6 0.573 39.6 0.456 39.6 0.477 39.6 0.552  Cohort #2 Male AVG ±SD 39.5 0.622 39.8 0.453 39.8 0.449 39.9 0.561 40.0 0.546 39.9 0.552  Cohort #2 Female AVG ±SD 39.8 0.675 39.7 0.711 39.5 0.624 39.9 0.596 39.9 0.555 39.6 0.354  Cohort #3 Male AVG ±SD 39.6 0.324 39.4 0.114 39.6 0.277 39.8 0.628 39.7 0.505 39.7 0.689  Cohort #3 Female AVG ±SD 39.9 0.641 39.7 0.308 39.6 0.379 39.6 0.306 39.6 0.402 39.4 0.279  Time -15 5 15 30 60 120  Cohort #4 Male AVG ±SD 39.7 0.383 39.6 0.294 39.7 0.261 39.7 0.320 39.8 0.297 39.9 0.320  Cohort #4 Female AVG ±SD 39.6 0.163 39.7 0.279 39.7 0.286 39.8 0.339 40.1 0.719 39.9 0.520  Cohort #5 Male AVG ±SD 39.4 0.153 39.4 0.000 39.4 0.100 39.4 0.115 39.5 0.208 39.4 0.100  Cohort #5 Female AVG ±SD 39.7 0.479 39.7 0.457 39.6 0.492 39.6 0.465 39.5 0.412 39.5 0.377  Cohort #6 Male AVG ±SD 39.5 0.585 39.5 0.603 39.5 0.418 39.4 0.548 39.5 0.575 39.4 0.402  Cohort #6 Female AVG ±SD 39.1 0.451 39.1 0.392 39.1 0.416 39.0 0.299 39.2 0.206 39.1 0.216  	
    201  

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