The purpose of this study was to determine the safety and pharmacokinetic profile of firocoxib in healthy neonatal foals. Foals are more sensitive to the side effects of nonsteroidal anti-inflammatory drugs, (NSAIDs), particularly due to immature renal clearance mechanisms and ulcerogenic effects on gastric mucosa. Firocoxib, a novel second generation NSAID, is reported to have reduced side effects due to its COX-2 selectivity. The pharmacokinetic profile of firocoxib in neonates has not been established, making reliable dosing difficult. We hypothesized that firocoxib given per os at the labeled dose to neonatal foals would be absorbed and not be associated with clinically significant adverse events. Seven healthy American Quarter Horse foals of mixed gender were administered 0.1mg/kg firocoxib orally q24h for nine consecutive days, commencing at 36h of age. Blood samples were collected for firocoxib analysis using high pressure liquid chromatography with fluorescence detection at 0 (dose #1 only), 0.25, 0.5, 1, 2, 4, 8, 16 and 24 hours after doses #1, 5 and 9. For all other doses (2, 3, 4, 6, 7 and 8) blood was collected immediately prior to the next dose (24 hour trough). Elimination samples (36, 48, 72, 96, 120 and 144 hours) were collected after dose #9. Safety was assessed via physical examinations, changes in body weight, gastroscopy, complete blood count, serum biochemistry and urinalysis. Firocoxib was rapidly absorbed following oral administration with minimal accumulation after repeat dosing. After the initial dose, an average peak serum concentration (Cmax) of 89.50 ° 53.36 ng/mL (mean ° SD) was achieved (Tmax) in 0.54 ° 0.65 hours. Steady state was obtained after approximately 4 doses and the average maximum concentration (Cavg) in serum was 39.1 ° 8.4 ng/mL. After the final dose, the mean terminal half-life (T½?») was 10.46 ° 4.97 hours. Firocoxib was not detected in plasma 72 hours after the final dose (<2ng/mL). Bioavailability could not be determined as currently, there is no accompanying intravenous dose of firocoxib for this age group to permit the calculation. No significant abnormalities were noted on blood work, urinalysis or gastroscopy. This study demonstrated that firocoxib is absorbed after oral administration in neonatal foals with no observable adverse effects after multiple doses. / Master of Science

Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is a common and potentially lethal infectious human disease. Rifampicin is a front line anti-tuberculosis drug usually prescribed in combination with isoniazid, pyrazinamide and streptomycin for a period of six to seven months. When given orally for the treatment of MTB, rifampicin exhibits low bioavailability. Recent attempts to increase bioavailability and decrease dosage of anti-tuberculosis drugs have focused on creating polymer coated rifampicin nanoparticles. The research effort presented in this thesis evaluates the formation, characterization and relative bioavailability of rifampicin loaded carboxymethylcellulose acetate butyrate (CMCAB) particles using two different formulation techniques. Multi inlet vortex mixer (MIVM) and manual spray drying techniques were used to form the rifampicin containing CMCAB particles. Characterization studies and analyses of particles revealed differences in particle sizes, shapes and drug loading between the different particle formulation techniques. In vivo pharmacokinetic studies in BALB/c mice indicate that a single dose of rifampicin laden CMCAB spray dried particle formulations are able to improve pharmacokinetic parameters including relative bioavailability of rifampicin compared to that of the free drug form at the same concentration. / Master of Science

No / Background: AQ4N (1,4-bis[[2-(dimethylamino)ethyl] amino]-5,8-dihydroxyanthracene-9, 10-dione bis-N-oxide dihydrochloride) is a prodrug which is selectively activated within hypoxic tissues to AQ4, a topoisomerase II inhibitor and DNA intercalator. Patients and methods: In the phase I study, 22 patients with oesophageal carcinoma received an i.v. infusion of AQ4N (22.5¿447 mg/m2) followed, 2 weeks later, by further infusion and radiotherapy. Pharmacokinetics and lymphocyte AQ4N and AQ4 levels were measured after the first dose. At 447 mg/m2, biopsies of tumour and normal tissue were taken after AQ4N administration. Results: Drug-related adverse events were blue discolouration of skin and urine, grade 2¿3 lymphopenia, grade 1¿3 fatigue, grade 1¿2 anaemia, leucopenia and nausea. There were no drug-related serious adverse events (SAEs). Three patients had reductions in tumour volume >50%, nine had stable disease. Pharmacokinetics indicated predictable clearance. Plasma area under the curve (AUC) at 447 mg/m2 exceeded AQ4N concentrations in mice at therapeutic doses and tumour biopsies contained concentrations of AQ4 greater than those in normal tissue. Tumour concentrations of AQ4 exceeded in vitro IC50 values for most cell lines investigated. Conclusions: No dose-limiting toxic effects were observed and a maximum tolerated dose was not established. Tumour AQ4 concentrations and plasma AUC at 447 mg/m2 exceeded active levels in preclinical models. This dose was chosen for future studies with radiotherapy.

Yes / Background: This study aims at characterizing the in vitro metabolism of cryptolepine using human and rat hepatocytes, identifying metabolites in rat plasma and urine after a single cryptolepine dose, and evaluating the single-dose oral and intravenous pharmacokinetics of cryptolepine in male Sprague Dawley (SD) rats. Methods: The in vitro metabolic profiles of cryptolepine were determined by LC-MS/MS following incubation with rat and human hepatocytes. The in vivo metabolic profile of cryptolepine was determined in plasma and urine samples from Sprague Dawley rats following single-dose oral administration of cryptolepine. Pharmacokinetic parameters of cryptolepine were determined in plasma and urine from Sprague Dawley rats after single-dose intravenous and oral administration. Results: Nine metabolites were identified in human and rat hepatocytes, resulting from metabolic pathways involving oxidation (M2-M9) and glucuronidation (M1, M2, M4, M8, M9). All human metabolites were found in rat hepatocyte incubations except glucuronide M1. Several metabolites (M2, M6, M9) were also identified in the urine and plasma of rats following oral administration of cryptolepine. Unchanged cryptolepine detected in urine was negligible. The Pharmacokinetic profile of cryptolepine showed a very high plasma clearance and volume of distribution (Vss) resulting in a moderate average plasma half-life of 4.5 h. Oral absorption was fast and plasma exposure and oral bioavailability were low. Conclusions: Cryptolepine metabolism is similar in rat and human in vitro with the exception of direct glucuronidation in human. Clearance in rat and human is likely to include a significant metabolic contribution, with proposed primary human metabolism pathways hydroxylation, dihydrodiol formation and glucuronidation. Cryptolepine showed extensive distribution with a moderate half-life. / Funded by Novartis Pharma under the Next Generation Scientist Program.

Aim. Baicalein is a bioactive flavonoid component isolated from the root of Scutellaria baicalensis, which has been used as a traditional Chinese medicinal herb for the treatment of inflammation for centuries. Although various pharmacological effects of baicalein have been demonstrated, only limited studies in rats reported pharmacokinetic of baicalein, which exhibited a low oral bioavailability due to extensive first-pass metabolism. In addition, no investigation on human oral absorption or metabolic kinetic profile was reported previously. The current project conducted a series of mechanistic studies aiming to elucidate the intestinal absorption, metabolism and disposition of baicalein. Since glucuronidation plays an important role in the first-pass metabolism of flavonoids including baicalein, additional studies on the relationship between human intestinal glucuronidation activities and chemical structures of flavonoids have also been performed. / Conclusion. Baicalein is well absorbed at intestine but subjected to extensive intestinal glucuronidation resulting in low oral bioavailability. The glucuronidation of baicalein is catalyzed by multiple UGT isozymes. The disposition of baicalein 7-O-glucuronide, the major metabolite of baicalein in vivo, is mediated by the MRP and OATP transporters. The nucleophilicity and stereo-conformation of -OH substituents are crucial for the intestinal glucuronidation of flavonoids. / Methods. For investigation on intestinal absorption, metabolism and disposition of baicalein, human Caco-2 cell monolayer model, rat in situ intestinal perfusion model, and in vitro metabolism model were employed in the present study. For the further investigation on the position preference on glucuronidation of flavonoids at human intestine, the in vitro rates of glucuronidation among seven commercially available mono-hydroxyflavones, namely 3-, 5-, 6-, 7-, 2'-, 3'- and 4'-mono-hydroxyflavones were determined and compared. / Results. The satisfactory permeabilities of baicalein obtained from both Caco-2 cell model and rat intestinal perfusion model indicated its potential good absorption at gastrointestinal tract. Therefore, absorption should not be the rate-limiting factor causing the low oral bioavailability of baicalein. However, extensive glucuronidation occurred in the rat intestine perfusion model with over 90% of baicalein being metabolized after intestinal absorption. Consistent findings were also observed in the in vitro enzyme kinetic studies of baicalein. The biotransformation of baicalein to baicalein 7-O-glucuronide was extensive in human liver microsome, human jejunum microsome, rat liver microsome, and rat jejunum microsome with intrinsic clearances (Vmax/Km) of 618, 446, 436, 298 mul/min/mg, respectively, which are orders of magnitude greater than those of most of western drugs that share the same metabolic pathway. Further enzyme kinetic studies using human recombinant glucuronosyltransferases (UGT) isozymes showed that UGT 1A1, 1A3, 1A8, 1A9, 1A7 and 2B15 were involved in the glucuronidation of baicalein with different kinetic profiles. Mechanistic studies on the disposition of baicalein 7-O-glucuronide formed from a rapid glucuronidation of baicalein in intestine demonstrated that this intracellularly formed glucuronide of baicalein could be actively extruded to both the apical and basolateral sides (the so called efflux) in Caco-2 cell model as well as rat intestinal perfusion model. It was also found that the efflux of the baicalein 7-O-glucuronide followed saturable enzyme kinetics and was effectively inhibited by multi-drug resistance associated proteins (MRP) and organic anion transporters (OATP) inhibitors. Further study on the relationship between flavonoid structures and glucuronidation activities using seven monohydroxyflavones demonstrated that the conjugation rates of 6- and 3'-monohydroxyflavones (HF) were much greater than those of 3-, 4'-, 7-, 2'-HF, while 5HF was the lowest. / Zhang Li. / "August 2006." / Advisers: Zhong Joan Zuo; Ge Lin. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1587. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 186-223). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

A following study on vancomycin demonstrated the differing pharmacokinetics during the course of a septic insult, day 2 pharmacokinetics differing from day 7. / An important study showed that some septic patients with "normal" serum creatinines can have very high creatinine clearances. It follows that drugs which are renally excreted will have high clearances and illustrates why many of the above patients had low serum levels of antibiotic, a reason why some ICU patients require different dosing to ward patients. / Due to the required fluid loading and inotropic use in septic patients, creatinine clearances and drug clearances are often raised. This results in low serum concentrations at the end of a standard dosing interval. / My beta-lactam antibiotic work has repeatedly demonstrated low serum levels at the end of the standard dosing interval. In view of beta-lactam time-dependent kill characteristics we designed a continuous infusion protocol which we validated in a follow-up paper. / The inflammatory response of infections involves endothelial damage and capillary permeability. With associated fluid shifts of severe sepsis and treatment thereof, the volume of distribution (Vd) of antibiotics that distribute into the extracellular space (aminoglycosides, glycopeptides) is high. Peak serum levels for these antibiotics are therefore lower than those found in non-critically ill and in normal volunteers. It is noteworthy that this change in Vd is not apparent with drugs that have good tissue penetration (e.g. ciprofloxacin). / This thesis is a compilation of 11 of my prospectively designed studies plus extracts from 5 published reviews, focusing on pharmacokinetic (PK) aspects of antibiotics in ICU patients, all published in internationally peer-reviewed journals. / Two large PK studies on ciprofloxacin (a drug that has excellent tissue penetration) designed to address possible PK differences over time, could not demonstrate this difference in adults nor in two groups of paediatric patients where differences in body water are significant. / Two papers investigated the pharmacokinetics of amicakin in adult and paediatric patients documenting the benefit of extended interval dosing. / We automatically assume that antibiotic prescribing data, collated from healthy volunteers and not so ill patients, can be transcribed into the Intensive Care Unit. This is not so. / Jeffrey Lipman. / "April 2005." / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1548. / Thesis (M.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 235-254). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / School code: 1307.

Methods. The intestinal absorption and metabolism of W and OA as well as the potential interactions among B, Wand OA were investigated at in vitro, in situ and in vivo levels. Various models were employed including Caco-2 cell monolayer model, in vitro enzymatic kinetics study, rat in situ single-pass intestinal perfusion model and in vivo pharmacokinetic study in rats. / Purpose. Scutellariae baicalensis Georgi is a medicinal plant widely distributed in Asia. Its dried root, Radix Scutellariae (RS), has been extensively used in Chinese and Japanese medicine. Six flavones including baicalein (B), wogonin (W), oroxylin A (OA) and their corresponding glucuronic acid conjugates (BG, WG, OAG) are the major bioactive components in RS. Our previous studies on B revealed an extensive first-pass metabolism during its absorption. Hence, it is expected that W and OA which have the similar structures as B, may share similar absorption and metabolic pathways as B. The present project aims to (1) establish an assay method for better quality control of RS; (2) provide further biopharmaceutic characterizations ofW and OA in RS; (3) investigate the potential pharmacokinetic interactions among B, Wand OA. / Results. Similar to B, Wand OA showed favorable permeability in both the Caco-2 cell and the rat in situ single-pass perfusion models. However, they experienced extensive first-pass metabolism, mainly in the form of glucuronidation. Intracellularly formed WG and OAG could be effluxed to both the apical side (lumen side) and basolateral side (mesenteric blood side) mainly by MRPs, which was confirmed by inhibition transport studies in Caco-2 cells and transfected MDCK cells. The glucuronidation rate of OA was higher than that of W, which was observed by enzymatic kinetics studies by sub-cellular fractions with intrinsic clearances (Vmax/K m, mul/min/mg) of 456 to 4170 for W and 509&sim;5038 for OA. UGT 1A9 was the most potent metabolic enzyme for hepatic glucuronidation, while UGTs 1A8 and 1AlO were responsible for the intestinal glucuronidation of W and OA. The in vivo rat pharmacokinetics studies showed that W and OA may be readily absorbed and extensively metabolized with no parent compound detectable in blood after oral administration of W and OA. A new metabolite of W was identified to be the glucuronic acid conjugate at 5-0H of W. After co-administration of B, W and OA, decreased formation of BG, WG and OAG was observed in in vitro enzymatic kinetics study. Further studies in absorption models of Caco-2 cell monolayer and rat in situ single-pass intestinal perfusion demonstrated the enhancement in absorption of B, W and OA and decrease of BG, WG and OAG after the co-administration of B, W and OA. The ultimate pharmacokinetics interaction study revealed that glucuronides were the predominant form in systemic circulation and the AUC of OAG significantly increased after co-administration of B, Wand OA. Conclusion: Similar to B, Wand OA may be well absorbed followed by extensive first-pass metabolism, which was mediated by various UGT isozymes. During absorption, the intracellularly formed WG and OAG were mainly effluxed by MRPs to both the lumen and mesenteric blood side of the intestine. Both in vitro and in situ models indicated that interactions among B, W and OA would lead to decreased glucuronidation and increased absorption of parent flavones. Due to extensive metabolism in vivo, only glucuronides appeared in systemic circulation after co-administration of B, W and OA in rats. The resulted increased systemic exposure of OAG indicated that the co-administration might lead to the enhancement of bioavailability for the studied flavones in the form of glucuronides. / Li, Chenrui. / Adviser: Zuo Zhong. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 201-236). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Objetivo: O objetivo do presente estudo foi descrever a relação entre as concentrações plasmáticas totais e livres tumorais do etoposídeo (ETO) e a inibição do crescimento do tumor observada em ratos Wistar portadores de tumor Walker- 256 (W256) utilizando a modelagem farmacocinética/farmacodinâmica (PK/PD). Métodos: Os procedimentos com animais foram aprovados no CEUA/UFRGS sob o número 22302. Os experimentos de farmacocinética foram realizados para determinar concentrações plasmáticas e livres em duas regiões do tumor sólido W256 através de microdiálise. Após a administração do ETO nas doses de 10 ou 20 mg/kg i.v. bolus em ratos Wistar portadores de tumor W256, amostras de sangue e microdialisado de tecido do centro e periferia do tumor foram coletadas simultaneamente, até 7 h pós-dose, para determinar o fator de penetração no tumor. Um método analítico por CLAE-UV foi desenvolvido e validado para quantificação do etoposídeo nas amostras de plasma e dialisado. Os experimentos de farmacodinâmica foram conduzidos em ratos portadores de tumor W256 que receberam ETO 5 e 10 mg/kg i.v. bolus uma vez ao dia por 8 e 4 dias, respectivamente. O volume dos tumores foram monitorados diariamente durante 30 dias. Análise não-compartimental dos dados de PK foi realizada no WinNonlin®. A modelagem dos dados PK e PK/PD foi realizada no Monolix®, utilizando abordagem populacional. Os dados PK/PD foram analisados usando o modelo Simeoni TGI modificado através da introdução de uma função Emax para descrever a relação nãolinear entre a concentração plasmática e tumoral e o efeito. Resultados e Discussão: O método por CLAE-UV foi desenvolvido e validado para quantificar as amostras de ETO em plasma e tecido. A penetração do ETO no tumor foi maior na periferia (61 ± 15 % e 61 ± 29 %) do que no centro do tumor (34 ± 6 % e 28 ± 11 %) após administração das doses 10 e 20 mg/kg, respectivamente (ANOVA, α = 0.05). Um modelo de 4 compartimentos compreendendo uma distribuição saturável (cinética de Michaelis-Menten) nos compartimentos tumorais a partir do compartimento central modelou simultaneamente os perfis de concentração-tempo do ETO em plasma e em ambas regiões do tumor. O modelo populacional PK/PD Simeoni TGI–Emax foi capaz de descrever o efeito antitumoral dependente do regime de administração do ETO utilizando concentrações totais plasmáticas ou livres no tumor, resultando em um maior k2max (potência máxima) para as concentrações livres (25,8 mL.μg-1.dia-1 - intratumoral vs. 12,6 mL.μg-1.dia-1 - plasma total). Conclusões: Os resultados mostram que a utilização das concentrações livres do fármaco no tumor para a modelagem PK/PD pode fornecer um melhor entendimento da relação farmacocinética e farmacodinâmica e melhoram a capacidade de previsão do modelo, considerando que a eficácia dos fármacos antineoplásicos no tratamento de tumores sólidos é dependente da capacidade do fármaco em se distribuir no tecido tumoral. / Objective: The aim of this study was to describe the relationship between total plasma and free interstitial tumor etoposide (ETO) concentrations and the drug tumor growth inhibition observed in a Walker-256 (W256) tumor-bearing Wistar rat model using the pharmacokinetic/pharmacodynamic (PK/PD) modeling. Methods: The experiments with animals were approved by CEUA/UFRGS (protocol number 22302). Pharmacokinetic experiments were conducted to determine total plasma and free intratumoral concentrations in two regions of W256 solid tumor by microdialysis. After administration of ETO 10 or 20 mg/kg i.v. bolus to W256 tumorbearing Wistar rats, blood and tissue microdialysate samples from tumor center and periphery were simultaneously collected up to 7h to determine the tumor penetration factor. An analytical HPLC-UV method was developed and validated for quantification of ETO in plasma and microdialysate samples. The pharmacodynamic experiments were conducted in W256 tumor-bearing rats that received ETO 5 or 10 mg/kg i.v. bolus every day for 8 and 4 days, respectively. Tumor volumes were monitored daily for 30 days. Non-compartmental analysis of PK data was performed in WinNonlin®. The PK and PK/PD modeling by population approach were performed using Monolix®. PK/PD data were analyzed using a modification of Simeoni TGI model by introducing an Emax function to describe the nonlinear relationship between tumor and plasma concentrations and effect. Results and Discussion: The HLPCUV method was developed and validated to determine plasma and tissue samples of ETO. ETO tumor penetration was higher in the tumor periphery (61 ± 15 % and 61 ± 29 %) than center (34 ± 6 % and 28 ± 11 %) following 10 and 20 mg/kg doses, respectively (ANOVA, α = 0.05). A 4-compartment structural model comprising a saturable distribution (Michaelis-Menten kinetics) into the tumor compartments from the central compartment simultaneously described the ETO concentration–time profiles in plasma and both tumor regions. The PK/PD population Simeoni TGI–Emax model was capable of describing the schedule-dependent antitumor effects of ETO using total plasma or free tumor concentrations obtained in a W256-tumor bearing Wistar rat model, resulting in higher k2max (maximal potency) for free concentrations (25.8 mL.μg-1.day-1 - intratumoral vs. 12.6 mL.μg-1.day-1 total plasma). Conclusions: The results showed that the use of free intratumoral drug concentrations in the PK/PD modeling can provide a better understanding of the pharmacokinetics and pharmacodynamics relationship and improve the forecasting ability of the models considering that the efficacy of antineoplastic drugs in the treatment of solid tumors is dependent on the drug ability to distribute into the tumor.

Bupivacaine ((R, S)-1-butyl-2-piperidylformo-2', 6'-xylidide), an anilide type local anaesthetic is manufactured in the standard racemic form and is widely used in the practice of regional anaesthesia. Despite its popularity as a local anesthetic, it has the potential to produce severe cardiotoxicity. Enantiomers, which are a pair of chiral isomers that are direct, nonsuperimposable mirror images of each other, vary in their chemical, pharmacological and toxicological profiles due to different stereospecific recognition in the body. Single enantiomeric drugs, when compared to racemic drugs, exert similar clinical effects but produce decreased risks of cardiac and neurotoxicity. This has led to the development of the single enantiomeric drugs ropivacaine ((S)-1-propyl-2-piperidylformo-2', 6'-xylidide) and levobupivacaine ((S)-1-butyl-2-piperidylformo-2', 6'-xylidide). Since local anaesthetics are extensively bound (>90%) to plasma protein in blood such as album and alpha1-acid-glycoprotein, it is only the free form of the flowing drug that can exert its pharmacological effects and are believed to be closely related to systemic toxicity. Although the safety and efficacy of these newer local anaesthetics have been ascertained in the literatures, but there are limited data on their pharmacokinetic profiles; thus it is envisioned that further pharmacokinetic trials would be required to elucidate their pharmacological and clinical effects. The aim of this thesis was to develop sensitive, reproducible and reliable methods of local anaesthetic assays to support such clinical trials. / The assays described in the thesis have been applied to numerous clinical research projects. Out of the various studies, the following will be discussed: Ropivacaine undergoes slower systemic absorption from the caudal epidural space in children than bupivacaine; Arterial and venous pharmacokinetics of ropivacaine with and without epinephrine after thoracic paravertebral block; Pharmacokinetics of levobupivacaine after thoracic paravertebral block. / The first method developed is the simultaneous determination of ropivacaine and bupivacaine in human plasma using high performance chromatography (HPLC). Most published methods of determining ropivacaine in human plasma use gas chromatography and a review of literature to date shows no data describing the use of HPLC to simultaneously determine both drugs. This is the first report describing a simple, isocratic, reversed-phase, liquid-liquid extraction procedure of high-performance liquid chromatographic method that allows the simultaneous detection of both local anaesthetics in one single injection. The chromatography was achieved using a reversed-phase chromatographic system with a Waters Novapak C18 column. 0.5 ml plasma was used for the sample preparation procedures. Bupivacaine and ropivacaine concentrations ranging from 10ng/ml to 3000 ng/ml and fixed amounts of pentycaine (internal standard) were spiked into the plasma samples for calculating the calibration graphs. Calibration graphs were linear over the range 10-3000 ng/ml (r=0.9978 for bupivacaine and r=0.9986 for ropivacaine). The within-day (intra-assay) coefficient of variation of the assay varied between 13.84% at 100 ng/ml, 1.84% at 500 ng/ml and 3.34% at 2000 ng/ml for bupivacaine; and 5.29% at 100 ng/ml, 1.38% at 500 ng/ml and 3.93% at 2000 ng/ml for ropivacaine. The between-day (inter-assay) coefficient of variation was 8.43% at 100 ng/ml, 4.06% at 500 ng/ml and 9.15% at 2000 ng/ml for bupivacaine, and 5.66% at 100 ng/ml, 4.40% at 500 ng/ml and 8.14% at 2000 ng/ml for ropivacaine. The limit of detection for both drugs was 10 ng/ml. / The fourth analytical technique describes the successful development of an ultrafiltration protein binding procedure to detect the free levels of the local anaesthetics in human plasma. Sample plasma was deposited in the ultrafiltration apparatus and ultrafiltrate containing the free local anaesthetics was forced thru a membrane under a fixed-angle rotor centrifugal force. Experiments were done to establish the optimum parameters for the ultrafiltration apparatus' binding capacities. The validated procedures use 0.5 ml plasma as the starting volume and it was deposited into the ultrafiltration apparatus. It was then subjected to 1750g centrifugal force for 20 minutes at centrifugal temperature of 37&deg;C. The resultant ultrafiltrate was processed according to the described LC-MS/MS method to detect the free local anaesthetic levels. / The second analytical methodology describes the assay of levobupivacaine in human plasma using HPLC. Calibration graphs relating peak height ratios and concentrations were linear over the range 10-3000 ng/ml (r=0.9995). The chromatography was achieved with an XTerra MS C18 column with the ultraviolet monitor set at 210 nm. The sample preparation steps were similar to the first analytical method, but with a different internal standard used. Precision and accuracy were assessed by performing analysis on replicate control plasma samples. The within-day (intra-assay) coefficient of variation of the assay varied between 4.25% at 50 ng/ml, 3.38% at 500 ng/ml, 3.76% at 1000 ng/ml and 3.14% at 2000 ng/ml. The between-day (inter-assay) coefficient of variation of the assay varied between 4.68% at 50 ng/ml, 4.94% at 500 ng/ml, 4.25% at 1000 ng/ml and 2.94% at 2000 ng/ml. The limit of detection was 10 ng/ml. / The third analytical methodology details the development and validation of a chiral analytical technique. This is the first report describing the development of a simple, isocratic, reversed-phase, liquid-liquid extraction procedure of a direct chiral method that allows the simultaneous detection of either free or total concentrations of bupivacaine enantiomers and ropivacaine in one single injection. It is also a novel technique to assay bupivacaine enantiomers with the use of vancomycin CSP column and liquid chromatography-mass spectrometry (LC-MS/MS) analysis, which achieved the lowest published detection limit with the lowest volume of plasma used. Calibration graphs were linear over the range 0.1-2000 ng/ml. Precision and accuracy were assessed by performing analysis on replicate control plasma samples. The within-day (intrassay) coefficient of variations of the assay for the drugs ropivacaine, levobupivacaine, dextrobupivacaine varied from 2.20% to 5.78%, 1.96% to 9.64%, 1.78% to 6.34%, respectively, for concentrations between 0.5 ng/ml to 2000 ng/ml. The between-day (interassay) coefficient of variations of the assay for the drugs ropivaciane, levobupivacaine, dextrobupivacaine varied from 3.66% to 9.61%, 3.18% to 8.34%, 2.22% to 10.59%, respectively, for concentrations between 0.5 ng/ml to 2000 ng/ml. The limit of detection was 0.05 ng/ml. / Wong Sum Yee April. / "November 2005." / Advisers: Manoj Karmakar; Tony Gin. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6370. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 207-217). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

<p>Cetirizine, an H₁-antihistamine, is prescribed for allergic disorders. It exists as a racemic mixture, with levocetirizine being the active enantiomer. The central nervous system side-effects of H₁-antihistamines are caused by their penetration into the brain. In this thesis the plasma pharmacokinetics, transport across the blood-brain barrier (BBB) and H₁ receptor occupancy of cetirizine enantiomers was investigated <i>in vivo</i> in guinea pigs. The transport across the BBB was quantified using the microdialysis technique. Stereoselective brain distribution was investigated by measuring both unbound and total concentrations in plasma and brain. The time aspects of the H₁ receptor occupancy of levocetirizine was studied in the brain and the periphery.</p><p>The plasma pharmacokinetics of cetirizine was stereoselective with clearance and volume of distribution of levocetirizine being approximately half that of dextrocetirizine. This was mainly due to the differences in plasma protein binding of the enantiomers. The stereoselectivity in brain distribution indicated by the partition coefficient K_p (total AUC ratio brain to plasma) was caused by stereoselective plasma protein binding. The transport across the BBB measured in this thesis by the unbound partition coefficient K_p,uu (unbound AUC ratio brain to plasma) was the same for the two enantiomers. Binding within the brain was also not significantly different. The H₁ receptor occupancy of levocetirizine in brain lagged behind the plasma concentrations whereas it was not delayed with respect to the brain concentrations. This indicates that the delayed brain H₁ receptor occupancy of levocetirizine is caused by a slow transport across the BBB.</p><p>In summary, the results of this thesis emphasize the importance of measuring both the unbound and total concentrations in blood and brain to characterize stereoselective brain distribution. The thesis also emphasize the importance of taking local brain pharmacokinetics into consideration in understanding pharmacokinetic-pharmacodynamic relationships of drugs with central activity.</p>