Master of Science / Clinical Sciences - Veterinary Medicine / Laurie A. Beard / Objective: To determine the pharmacokinetic properties of oral pergolide in normal mares. Animals: 6 horses, 3-17 years of age, 355-582 kg Procedures: In a randomized, cross over design six healthy adult female horses received pergolide (PO) 0.01mg/kg or placebo after 8 hours of fasting. Samples were taken over a period of 6 day for each portion of the study (treatment or placebo) with a two week minimum wash out period between study periods. Quantification of pergolide concentration was determined by UPLC-MS. Quantification of α-MSH was determined by radioimmunoassay validated for horses. Quantification of ACTH concentration was determined by chemiluminescent enzyme immunoassay. Results: Pergolide was detected in all treated horses. The relatively short time to peak concentration (0.5 hours) indicates a rapid absorption. Mean maximum concentration measured was 4.05 ng/ml + 2.02 with a median time to maximum concentration being 0.415 hours (range:0.33-1.0). The mean half life of pergolide was determined to be 5.86 hours + 3.42. Lower limits of quantitation for the UPLC-MS assay was 0.5 ng/ml. α-MSH results were evaluated using a multiple analysis of variance assay for repeated measures comparing treatment, time, and period. There was a significant treatment to period effect with p=0.02. The effect of period appears to be more significant (p=0.06) compared to the effect of treatment (p=0.77). No effect from pergolide was noted on ACTH concentrations. Conclusions and Clinical Relevance: Horses appear to absorb and eliminate pergolide more rapidly than previously expected. Based on this pharmacokinetic data the dosing strategies of pergolide may need to be altered. However, assay sensitivity does need to be improved prior to recommendations being made.

Part I. Following a general historical review of the development of drug targeting, critical evaluations were made of current targeted drug delivery systems. Based on the results shown by previous studies, magnetic albumin microspheres containing adriamycin is one of the most promising targetable delivery systems for the treatment of solid tumors. It was also apparent that the pharmacokinetics of drugs associated with magnetic albumin microspheres had not been determined. A systematic study of the multiple variables involved in albumin microsphere preparation was completed to identify to what extent these variables affected the microsphere size distribution. The results of this investigation led to an optimal method of microsphere preparation. Information obtained from the above studies was applied to the production of magnetic albumin microspheres containing adriamycin suitable for in vivo use. The problems of separation and quantitation of adriamycin and adriamycinol in biological matrices were investigated using ion-pairing high pressure liquid chromatography. An optimized chromatographic system was presented for the analysis of these compounds in rat serum and tissues. The disposition of adriamycin following administration as magnetic albumin microspheres and as a solution was studied by monitoring adriamycin concentrations in multiple rat tissues for forty-eight hours after administration. The magnetic dosage form was targeted to a predefined tail segment with a magnetic field strength of 8000 G applied for 30 min after dosing. A physiological pharmacokinetic model was used to describe the disposition of adriamycin after both dosage forms. The model developed following adriamycin administration as a solution served as the foundation for the model for adriamycin when it was administered as the magnetic dosage form. Part II. The present investigation was designed to characterize the kinetics of ibuprofen in plasma and synovial fluid, which in the past, has been flawed by inadequate study protocols. After administration of a single dose and at steady-state, ibuprofen concentrations were measured simultaneously in plasma and synovial fluid obtained from eight patients with rheumatoid arthritis. The extent of accumulation of ibuprofen in each fluid was determined. The degree of ibuprofen protein binding in plasma and synovial fluid was also determined and related to its kinetic behavior.

Submitted in fulfillment of the requirements of the degree of Master of Applied Science in Chemistry, Durban University of Technology, 2015. / Human Serum Albumin (HSA) predominantly found in the blood plasma proteins, acts as a carrier for many drugs. In the present work binding interactions of eight arylpropionate non-steroidal anti-inflammatory drugs (NSAIDs) were studied with Human Serum Albumin HSA using Capillary Electrophoresis (CE) under physiological conditions. The concentration of HSA was kept constant (525 μM) whereas the drug concentrations were varied between 50-300 μM in each case. The Frontal analysis (FA) and Capillary Zone Electrophoresis (CZE) modes of CE were applied together with a mathematical modelling of the experimental results with a view to obtaining pharmacokinetic properties of each drug. The binding order of the drugs to HSA were established with the three methods together with the mathematical approach. Our studies revealed the presence of more than one binding sites for some of the available drugs. Additionally, molecular docking studies were conducted to establish the binding conformations of drugs in the binding pocket of the HSA. A very good correlation between the computed binding energies (docking) and the experimental binding constants were observed throughout this study. The logK values for all eight drugs were ranging from 3.37 - 4.56 for FA, 3.16 – 4.39 for CZE, and 3.48 – 5.30 for computational studies. / M

Part I. Seven healthy male subjects each received on a weekly basis placebo or 10, 20, 40, 80, or 160 mg oral propranolol (P) doses q.i.d. The effect of P on resting heart rate (HR) and the HR response to the Valsalva's maneuver, tilt, isoproterenol (ISO), and exercise were measured. The results indicate that the resting HR and the Tachycardiac response to Valsalva and tilt cannot be used to estimate beta blockade (BB). Although P serum levels correlated well (r² = 0.80) with the ISO dose ratio minus one, ISO challenges appear clinically inappropriate. Reduction in exercise tachycardia correlated best with P serum levels (r² = 0.89). In patients on P therapy, in which exercise would be contraindicated, there appears to be no reliable and safe method of clinically documenting BB. Part II. The parmacokinetics of intravenous P were studied in calves before and after biochemical induction of thyrotoxicosis. The beta adrenergic response to P was measured in both euthyroid and thyrotoxic (T) animals at steady-state serum levels of P by administration of ISO. No pharmacokinetic differences were detected between animal groups; however, T calves displayed a markedly different pharmacologic response to P. On the average, 2-9 times higher serum levels of P were required to facilitate BB in the T calves. These results suggest that in the calf model, thyrotoxicosis induces a decreased sensitivity to P independent of laterations in P's disposition. Part III. The aim of this study was to find a digitalis glycoside (DG) with a t(½) shorter than that of digitoxin (DT), but similar to that of digoxin, and whose disposition characteristics are not influenced by alterations in renal function, as is the case with digoxin. Consequently, the pharmacokinetics of two metabolites of DT, digitoxigenin-bisdigitoxoside (BIS) and digitoxigenin-monodigitoxoside (MONO), were compared to those of DT in a dog model. In normal dogs, appreciable differences were found between the systemic clearance (CL) of DT and the CL of either of the two other DG's. These differences in CL were primarily responsible for the 2.0 and 3.5 fold decrease seen in the t(½)'s of BIS and MONO, respectively. Renal disfunction did not influence the pharmacokinetic parameters of any of the DG's studied. These findings in the dog model suggest that BIS or MONO may provide a pharmacokinetic advantage over DT.

Angiogenesis is a fundamental driver of tumor biology and many other important aspect of human health. Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI) has been shown to be a valuable biomarker for the indirect assessment of angiogenesis. However, DCE-MRI is very specialized technique that has limitations. In this dissertation new models and contrast agents to address some of these limitations are presented. Chapter 1 presents an introduction to DCE-MRI, the rationale to asses tumor biology with this technique, the MRI pulses sequences and the standard pharmacokinetic modeling used for the analysis of DCE- MRI data. Chapter 2 describes the application of DCE-MRI to asses the response to the hypoxia-activated drug TH-302. It is shown that DCE-MRI can detect a response after only 24 hours of initiating therapy. In Chapter 3, a new model for the analysis of DCE-MRI is presented, the so-called Linear Reference Region Model (LRRM). This new model improves upon existing models and it was demonstrated that it is ~620 faster than current algorithms and 5 times less sensitive to noise, and more importantly less sensitive to temporal resolution which enables the analysis of DCE-MRI data obtained in the clinical setting, which opens a new area of study in clinical MRI. Chapter 4 describes the extension of the LRRM to estimate the absolute permeability of two fluorinated contrast agents; we call this approach the Reference Agent Model (RAM). In order to make this new model an experimental reality, a novel pulse sequence and contrast agents (CA) for ¹⁹F MRI were developed. Two contributions to the field of DCE-MRI are presented in this chapter, the first simultaneous ¹⁹F-DCE-MRI detection of two fluorinated CA in a mouse model of breast cancer, and the estimation of their relative permeability. RAM eliminates some of the physiological variables that affect DCE-MRI, which may improve its sensitivity and specificity. Finally, new potential applications of LRRM and RAM are discussed in Chapter 5.

No description available.

The aims were to critically assess methodologies relevant to PK studies tailored to the needs of children. In view of the significant challenges associated with pain control in children paracetamol and ketorolac were selected for the study. Methods: Two bioanalytical methods were developed to measure paracetamol and enantiomers of ketorolac in paediatric samples, to support PK studies of IV paracetamol and ketorolac in children less than 16 years old. All studies had ethical approval and clinical trial authorisation. Results: The developed bioanalytical methods were reliable, requiring only 30 μl of blood for paracetamol, and 50 μl of plasma for ketorolac. Paracetamol concentrations from finger-prick samples in the first hour post dose were greater than from venous samples (349% at 15, 72% at 30, and 9.3% at 60minutes). There were no differences in median CL (0.41, 0.31 and 0.37 1h^-1kg^-1), V_d (0.90, 0.95 and 0.90 1/kg) and t_1/2 (1.7, 2.2 and 1.6 h) among the respective age groups 2-5, 6-10 and 11-15 years. All PK parameters were different for R- and S- ketorolac, with V_d and CL of R- being lower than those of S- (0.12 vs. 0.17 1/kg; 0.017 vs. 0.049 1/h/kg). The median half-life of R- was longer than that of S- (5.0 vs. 3.1 h, <i>P =</i> 0.043). Conclusions: Bioanalytical method did not limit PK trials in children. Sampling method affected PK parameter estimation, and prolonging cannula patency was the most challenging procedure during blood sampling. The PK of IV paracetamol was similar in children 2-15 years old, and the enantiomers of ketorolac were found to have different PKs in children.

This research developed and validated QSPKR models for predicting in-vivo human, systemic biologically relevant PK properties (i.e., reflecting the disposition of the unbound drug) of four, preselected, pharmacological classes of drugs, namely, benzodiazepines (BZD), neuromuscular blocking agents (NMB), triptans (TRP) and class III antiarrhythmic agents (AAR), as well as PK allometric scaling (PK-AS) models for BZD and NMB, using pertinent human and animal systemic PK information (fu, CLtot, Vdss and fe) from published literature. Overall, lipophilicity (logD7.4) and molecular weight (MW) were found to be the most important and statistically significant molecular properties, affecting biologically relevant systemic PK properties, and the observed relationships were mechanistically plausible: For relatively small MW and lipophilic molecules, (e.g., BZD), an increase in logD7.4 was associated with a decrease in fu, an increase in Vdssu and CLnonrenu, suggesting the prevalence of nonspecific hydrophobic interactions with biological membranes/plasma proteins as well as hepatic partitioning/DME binding. Similar trends were observed in fu and Vdssu for intermediate to large MW, hydrophilic molecules (e.g., NMB). However, although similar trends were observed in fu and Vdssu for relatively hydrophilic, intermediate MW molecules (e.g., TRP), and a heterogeneous class (e.g., Class III AAR), logD7.4 and MW were found to be highly correlated, i.e., the indepdendent effects of logD7,4 and MW cannot be assessed NMB, TRP and Class III AAR show mechanistically diverse clearance pathways, e.g., hepatobiliary, extrahepatic, enzymatic/chemical degradation and renal excretion; therefore, effects of the logD7.4 and/or MW are note generalizable for any of the clearances across classes. PK-AS analyses showed that Vdssu and Vdss scaled well with body weight across animal species (including humans) for BZD. Overall, within the limitations of the methods (and the sample size), ‘acceptable’ predictions (i.e., within 0.5- to 2.0-fold error range) were obtained for Vdssu and Vdss for BZD (and fu correction resulted in improvement of the prediction); however, none of the CLtot predictions were acceptable, suggesting major, qualitative interspecies differences in drug metabolism, even after correcting for body weight (BW). NMB undergo little extravascular distribution owing to their relatively large MW and charged nature, and, as a result, a high percentage of acceptable predictions was obtained for Vdss (based on BW). Similarly, the prediction of CLren (based on BW and glomerular filtration rate, GFR) was acceptable, suggesting that NMB are cleared by GFR across species, and there are no interspecies differences in their tubular handling. On the other hand, CLtot (and/or CLnonren) could not be acceptably predicted by PK-AS, suggesting major differences in their clearance mechanisms across animal species.

Master of Science / Department of Clinical Sciences / Warren L. Beard / Pharmacokinetics of Intramuscular Morphine in the Horse Elizabeth Devine, DVM; Butch KuKanich, DVM, PhD, DACVCP; Warren Beard, DVM, MS, DACVS Objective - To determine the pharmacokinetics of morphine after intramuscular administration in a clinical population of horses Design – Prospective, clinical study Animals – Pilot study included 2 normal horses and the clinical study included 75 horses Procedures – Morphine was administered at 0.1mg/kg, IM and 2-3 blood samples were obtained from each horse at various times from 0-9 hours after administration. Plasma morphine concentrations were measured using liquid chromatography and mass spectrometry. Results – Data was analyzed using a naïve pooled pharmacokinetic model. The half-life for the elimination phase was approximately 1.5 hours, the volume of distribution (per bioavailability) was approximately 4.5 L/kg and the clearance (per bioavailability) was approximately 35 mL/kg/min. The peak plasma concentration was 21.6 ng/mL and occurred approximately 4 minutes after administration. Plasma concentrations of morphine were below the limit of quantification by 7 hours in 74 horses. Conclusions and Clinical Relevance – The relatively short half-life of morphine indicates the need for frequent dosing to maintain targeted plasma concentrations. Adverse effects were uncommon in this study and morphine was well tolerated at a dose of 0.1 mg/kg, IM. Morphine may be a useful adjunctive therapy in painful horses, but the variable plasma concentrations suggest the dose and dosing interval may need to be adjusted to the individual patient’s response.

The ethical and practical issues of obtaining a blood sample pose a significant challenge to performing pharmacokinetic studies in children, infants and neonates. Dried blood spot analysis, based on the collection of a micro blood sample has potential to overcome these difficulties. There are at present a limited number of reports on the utility of dried blood spot analysis in clinical pharmacokinetic studies. The studies described in this thesis were undertaken to investigate the accuracy and precision of dried blood spot sampling coupled with mass spectrometry detection for drug quantification, and clinically validate the robustness and feasibility of this technique for pharmacokinetic studies in preterm neonates. Dried blood spot methods were developed for application to pharmacokinetic studies of test drugs dexamethasone and caffeine. Investigations were focused on the blood collection system, analyte recovery and optimisation of the detection system. In-vitro validation results indicated developed methods were precise, accurate and selective in accordance with the Food and Drug Administration regulatory guidelines on the assessment of bioanalytical methods. Results were not significantly affected by small variations in the blood volume spotted or the presence of petroleum jelly, which is often used on the sampling site during capillary blood collection in neonates. Variability in haematocrit was determined to be the single most important factor affecting assay accuracy. Stability assessments by comparison with freshly prepared samples verified the suitability of sample drying, storage and post sample extraction conditions. An investigation of method transferability between different analytical instruments was undertaken with caffeine to provide an assessment of the robustness of dried blood spot analysis. Results generated from a single and triple quadrupole mass spectrometer were comparable with an expected lower limit of quantification with the latter technique most likely due to a greater ionisation and detection efficiency. Intravenous dexamethasone pharmacokinetics was determined in 5 preterm neonates receiving treatment for chronic lung disease. Individual pharmacokinetic analyses were performed using a one compartment model to estimate primary pharmacokinetic parameters, clearance (mean, 0.18 l/h/kg) and volume of distribution (mean, 1.33 l/kg). The whole blood derived mean estimates were similar to previous plasma clearance and volume estimates of 0.14 l/h/kg and 1.91 l/kg, respectively reported in neonates (n=7). This highlights the potential for dried blood spot analysis as an alternative to conventional plasma based methods for dexamethasone dose optimisation studies in neonates. The population pharmacokinetics of oral / intravenous caffeine was determined in 67 preterm neonates. A one compartment model was used to describe the blood concentration-time data. Model evaluation using a bootstrapping technique confirmed the robustness and stability of the developed model. Pharmacokinetic parameters derived from dried blood spot drug measurements were estimated with precision (relative standard error &lt; 10%) and were comparable to estimates of plasma clearance (mean, 7.3 vs. 7.0 ml/h/kg) and volume of distribution (mean, 593 vs. 851 ml/kg) from a previous population study in neonates (n=110). Weight and postnatal age were the most influential covariates in the clearance model which is in agreement with previous population studies. These results demonstrate that dried blood spot analysis is a practical technique, with significant potential as a robust method for use in clinical pharmacokinetic studies in vulnerable populations such as preterms. Haematocrit related effects on paper will need to be accounted for if this potential is to be realised. Further investigations to determine the reproducibility of capillary blood sampling in neonates and the impact of using blood drug measurements on pharmacokinetic parameter estimation will be necessary before widespread use of the technique is possible.