A 'steady-state' constant plasma concentration paradigm was used to assess: a) whether rapid acute tolerance to the central nervous system effects occurred following administration of three centrally acting drugs and, b) the profiles of psychomotor impairments obtained under these constant conditions. The issue of differential test sensitivites at steady-state was also addressed. The drugs used were chlormethiazole, ethanol and nitrous oxide. These were administered by the intravenous, oral and inhalation routes respectively. Central nervous system effects were assessed using a battery of standard and new psychological tests and subjective measures which were first assessed for reliability. In terms of cognitive and psychomotor performance, there was no evidence of acute tolerance to chlormethiazole or to ethanol at steady-state. There was, however, evidence of acute tolerance to some peripheral effects of chlormethiazole, and to some of the subjective effects of nitrous oxide. The patterns of effects were consistent with the view that the drugs had broadly the same profile of effect on the tests used, but at different points on the same basic dose response curve of impairment. The exception was critical flicker frequency which was less affected than expected with chlormethiazole (a hypnotic) and nitrous oxide. The nitrous oxide data suggested that some of the dose-response curves for different processes may cross over. Thus body sway which was substantially impaired with all three drugs, was one of the least sensitive tests at the lower concentrations of nitrous oxide. Tapping, on the other hand, was sensitive at the lower concentrations, but the magnitude of impairment was smaller than for other tests at the higher concentrations.

The intravesical administration of <i>Bacillus Calmette Guerin</i> for the treatment of transitional cell carcinoma of the bladder is the most effective immunotherapy for any solid human malignancy. Despite this awesome accolade relatively little is understood of its mechanism of action. This study details the <i>in vitro</i> interaction between IL-2 activated lymphocytes and tumour cells, the effect of cytokines produced as a result of immunotherapy on tumour cells and the relationship of these findings to the situation <i>in vivo</i>. Bladder cancer cells were not found to be susceptible to NK cell activity but were found to be differentially susceptible to IL-2 activated lymphocytes. No correlation was evident between the histopathological grade of the tumour. The interaction between these cells was observed to involve intimate contact and the tumour cells were found to constitutively express either ICAM-1 or ICAM-2. The expression of these cell adhesion molecules correlated significantly with the sensitivity of the tumour cells to LAK mediated cytolysis. Following BCG therapy a variety of cytokines including IFNγ and TNFα are detected in the urine. When bladder cancer cells were cultured in the presence of recombinant IFNγ and TNFα an increase in the levels of ICAM-1 expression was observed. The optimal stimulation was found after 24 hours culture with 100Uml<SUB>-1</SUB> IFNγ, whilst TNFα stimulated to a lesser extent. Culture in the presence of both cytokines was observed to synergistically induce or augment ICAM-1 expression. Following culture with IFNγ, the tumour cells displayed increased susceptibility to LAK activity, this was significantly correlated with increased ICAM-1 expression. The levels of tumour cell response to IFNγ could not be correlated with either the abundance or affinity of specific receptors as determined by Scatchard analysis. Thus investigations were initiated into the events down-stream of the ligand-receptor interaction. Monoclonal antibodies to ICAM-1, decreased the sensitivity of tumour cells to LAK activity. However, monoclonal antibodies to LFA-1 (the ligand for ICAM-1) further blocked the action of LAK cells.

Agonist-induced contraction of airways smooth muscle is mediated by phosphoinositide hydrolysis and the production of the second messenger inositol 1,45-trisphosphate (Ins(1,4,5)P<SUB>3</SUB>). Muscarinic receptor-stimulation of bovine tracheal smooth muscle (BTSM) results in a transient increase in Ins(1,4,5)P<SUB>3</SUB> mass despite a sustained, non-desensitising hydrolysis of its precursor phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P<SUB>2</SUB>). Hence the rapid metabolism of Ins(1,4,5)P<SUB>3</SUB> appears to be the major regulator of Ins(1,4,5)P<SUB>3</SUB> levels under agonist-stimulated conditions. A model system has been developed to facilitate detailed study of the pathways involved in this complex metabolism. BTSM slices were labelled to equilibrium with <I>myo</I>-[<SUP>3</SUP>H]inositol in the presence of agonist and subsequent carbachol (CCh)- or histamine (Hist)-stimulations carried out in the presence of lithium ions to block InsP<SUB>1</SUB> breakdown. A delayed accumulation of [<SUP>3</SUP>H]Ins1/3P and [<SUP>3</SUP>H]Ins4P was observed under agonist-stimulated conditions. Moreover, there was no demonstrable phosphoinositide hydrolysis either following membrane-depolarisation, or secondary to a physiologically relevant increase in intracellular calcium in this thesis. The model therefore provides an appropriate system for the study of receptor-stimulated PtdIns(4,5)P<SUB>2</SUB>-derived Ins(1,4,5)P<SUB>3</SUB> metabolism. Cell-free experiments confirmed that Ins(1,4,5)P<SUB>3</SUB> is metabolised primarily by two different pathways - a 3-kinase and a 5-phosphatase pathway - which yield mutually exclusive products. H.P.L.C. separation of the individual [<SUP>3</SUP>H]inositol polyphosphate (InsPP) isomers accumulating in BTSM slices enabled the 3-kinase and 5-phosphatase metabolites to be quantified, and facilitated the determination of flux of the inositol headgroup through these two pathways. The pattern of Ins(1,4,5)P<SUB>3</SUB> metabolism varies during the lifetime of the agonist-stimulated response. The 5-phosphatase enzyme is highly dominant especially at early time-points following agonist-stimulation, whilst the 3-kinase becomes increasingly important at later time-points.

Patients with renal failure react inappropriately to many drugs and have an increased incidence of side effects which may be due to changes in drug absorption, distribution, metabolism and excretion. Active or inactive polar drug metabolites which are normally excreted in the urine will accumulate and patients with end stage disease may depend completely on haemodialysis or continuous ambulatory peritoneal dialysis (CAPD) for their elimination. Despite several single dose studies little information exists about the disposition of paracetamol and frusemide during chronic dosing in patients with renal failure including those maintained on dialysis and this has been investigated further in the present study. Six patients with end stage disease maintained on CAPD were given 1 g of oral paracetamol. Absorption was normal but plasma concentrations of the glucuronide and sulphate conjugates were greatly increased with little change during the observation period. The extraction capacity of the peritoneal membrane was low and the peritoneal clearance was < 7ml.min<SUP>-1</SUP> for paracetamol and these conjugates. This disposition of paracetamol was then compared in 6 healthy volunteers and 6 conservatively managed patients with chronic renal failure taking 1 g 3 times a day for 10 days. Before dosing, the daily plasma concentrations of unchanged paracetamol were significantly higher in the patients (3.1 ± 0.6 versus 1.1 ± 0.3 mg.l<SUP>-1</SUP>) suggesting enterohepatic recycling with regeneration by hydrolysis of the conjugates and reabsorption of the parent compound. There was marked accumulation of the glucuronide conjugate (87.0 ± 69.0 versus 3.0 ± 0.5 mg.l<SUP>-1</SUP> in the volunteers) which was dependent on the severity of the renal failure. The concentrations of the sulphate conjugate (25.0 ± 19.0 mg.l^-1) did not accumulate as predicted possibly due to depletion of inorganic sulphate. Patients with end stage renal failure maintained on haemodialysis were also treated with a similar regime of paracetamol. Neither the glucuronide nor sulphate conjugate reached the predicted mean plasma concentrations of 569 ± 150 and 434 ± 92 mg.l^-1 and extraction ratios of paracetamol and its conjugates were less than 50%.

Prostaglandin (PG) E<SUB>2</SUB> has been shown to act on at least three distinct receptor subtypes, designated EP<SUB>1</SUB>, EP<SUB>2</SUB> and EP<SUB>3</SUB>, with recent evidence suggesting the possibility of a fourth EP-receptor subtype. Whilst good antagonists are not available, a range of PGE analogues of differing selectivity for the three receptor subtypes may be used to determine which is effective in a given system. The activity of these analogues has not yet been assessed on the putative EP<SUB>4</SUB>-receptor containing preparation. This research has involved the use of such compounds in an <i>in vivo</i> model of rabbit-skin inflammation, and biochemical studies on both platelets and cultured macrophages, to determine the subtype(s) of PGE receptor mediating the pro-inflammatory, pro-aggregatory, and anti-inflammatory effects, respectively, of PGE<SUB>2</SUB>. Whilst PGE<SUB>2</SUB> alone had little effect on rabbit skin inflammation, potentiation of vascular permeability induced by mediators of inflammation, such as bradykinin (BK) and FMLP was observed. The potentiation has been attributed to the vasodilator activity of PGE<SUB>2</SUB> which is typically mediated via the EP<SUB>2</SUB>-receptor subtype. However the finding that compounds with both EP<SUB>2</SUB>- and EP<SUB>3</SUB>-receptor activity were the most active, suggested that vasodilatation was not the sole mechanism of the potentiation. A comparison of the ability of PGE<SUB>2</SUB> and the stable PGI<SUB>2</SUB> analogue, cicaprost, to induce vasodilatation and potentiate the responses to both BK and FMLP, was consistent with this suggestion. It has since been proposed that there is an initial EP<SUB>3</SUB>-receptor mediated, dilatation-independent component to the potentiation of BK by PGE<SUB>2</SUB>.

Primary afferent neurons with perikarya in the dorsal root ganglia (<i>drg</i>) mediate the transmission of sensory information from the periphery and viscera to the spinal cord. A number of neuropeptides, including substance P (SP), somatostatin (SS) and calcitonin gene related peptide (CGRP) have been localised in discrete but often overlapping subpopulations of <i>drg</i> neurons. These neuropeptides have been implicated in nociception and neurogenic inflammation, both processes which can be influenced by steroid hormones. Whilst steroid hormones have been shown to regulate synthesis of a number of neuropeptides including SP, SS and CGRP in the central nervous system, it is not known if they influence neuropeptides in the <i>drg</i>. In this thesis I have investigated the regulation of SP, SS and CGRP by steroids <i>in vivo</i> and in dissociated primary cultures of adult <i>drg</i> neurons. The SP, SS and CGRP content of <i>drg</i> from C1 to L6 was determined and a differential distribution of the three neuropeptides was found. <i>Drg</i> wet weight and SP content were greatest in <i>drg</i> from the cervical (C6 - T1) and lumbar (L4 - L6) enlargements. CGRP content tended to be greater in <i>drg</i> from more caudal areas (T10 - L6) and SS content was uniform in most <i>drg</i> but was notably lower in <i>drg</i> from area C5 - C7. The regulation of neuropeptides in the <i>drg</i> by two classes of steroid, adrenal steroids and androgens, was investigated. Adrenalectomy (ADX) of adult male Wistar rats increased SP and CGRP and decreased SS content of cervical <i>drg</i>. The effects of ADX were reversed by subcutaneous implantation of pellets containing corticosterone (B) or by daily subcutaneous injection of dexamethasone (DEX). The neuropeptide content of <i>drg</i> was not regulated by androgens; no significant change in the SP, SS or CGRP contents of <i>drg</i> were found following castration or administration of supraphysiological testosterone.

Three ovarian carcinoma continuous cell lines, A2780, 2780AD and 2780CP, were found to have a stable, 80-fold, range of cisplatin sensitivity, using a modified MTT chemosensitivity assay. This experimental model was used to investigate various theoretical mechanisms of platinum resistance. Inductively coupled plasma mass spectrometry (ICPMS), a highly sensitive technique for platinum analysis, was used to develop a new assay for the direct measurement of intrastrand platinum-DNA adducts. Highly platinated calf thymus DNA was disaggregated enzymatically; nucleotides and platinum-containing oligonucleotides were separated by HPLC using an anion exchange column. Purified adduct standards were used to calibrate the chromatogram; platinum analysis of eluate fractions revealed two peaks coinciding with the eluate positions of the two major intrastrand adducts. Electrothermal vaporisation, a modification of ICPMS, was used to improve analytical sensitivity prior to the direct investigation of intrastrand adducts in this cellular model of induced platinum resistance. Accumulation of platinum, both intracellular and DNA-bound, was measured by ICPMS; a linear relationship was observed between intracellular platinum levels and chemosensitivity for all three cell lines. Platinum accumulation was reduced in both resistant sublines, although to a similar degree in each. Uptake did not appear to be energy-dependent; nor did the P glycoprotein efflux pump appear to be a significant factor. Induced cisplatin resistance was found to be associated with elevated levels of reduced glutathione (GSH). The inhibitor of glutathione synthesis, buthionione sulfoxidine(BSO), was used to achieve up to 100% reduction in GSH levels; however no effect on cisplatin sensitivity was observed under these experimental conditions.

The benefits of thrombolytic therapy in acute myocardial infarction are established, yet there remain important questions, including the safe interval for effective readministration of the streptokinase-containing thrombolytic agents, and how to expedite the administration of therapy and the restoration of coronary patency. The work in this thesis addresses some aspects of these questions. The administration of thrombolytic agents by bolus injection has practical attractions. The pharmacokinetic properties of streptokinase (SK) and anistreplase in their standard administration regimens are compared, and demonstrate the significantly earlier, higher plasma concentrations and longer half-life achieved by bolus administration of anistreplase. Domiciliary administraiton reduces the delay to therapy. The feasibility of this approach was assessed comparing general practitioners' assessment of 139 patients, with and without electrocardiographs, with the coronary care unit diagnosis and the prescription of thrombolytic therapy. Domiciliary assessment is insufficiently reliable to recommend routine use of thrombolytic therapy at home at the present time. The significance of antibodies to SK-containing thrombolytic agents is largely unknown. Pretreatmet SK resistance titre and anti-SK IgG concentrations were measured in 128 patients with acute myocardial infarction treated with SK or anistreplase. A significant minor negative influence of SK resistance titre on coronary patency was observed. The haemodynamic responses to these agents were observed in detail, and blood pressure falls found to be usually short-lived and not require specific intervention. An association was sought between hypotensive episodes and markers of immunological resistance, markers of thrombin activity, and plasma viscosity. No relationship was found, refuting their implication in the hypotensive mechanism. The time course of the development of changes in SK resistance titre and anti-SK IgG concentration were documented in detail in the same patients over 30 months. Both indices peaked at 2 weeks following therapy, and declined slowly, with 50 & 58% of the population returning to within two standard deviations of pretreatment levels within 2 years. The functional sequelae of these antibody responses were studied in vitro using a pooled clot lysis assay. These data demonstrated near complete inhibition of lysis up to 9 months, with 75% recovery at 30 months.

The administration to normal men of supraphysiological doses of testosterone causes a profound inhibition of spermatogenesis and is currently being investigated as a method of male contraception. Azoospermia, however, is achieved in only 50-70% of men, the remainder maintaining a very low rate of spermatogenesis. The object of these studies was to investigate the biochemical basis for the maintenance of spermatogenesis in the oligozoospermic group, and in particular to investigate the hypothesis that the activity of the enzyme 5α-reductase, which converts testosterone to the more potent androgen dihydrotestosterone (DHT), is increased in these men either constitutionally or as a result of treatment. Thirty-three normal men were recruited to a clinical trial of hormonal male contraception. After a baseline period, subjects were administered 200 mg testosterone oenanthate i.m. weekly. Semen samples were analysed at 4 weekly intervals. When the sperm density had fallen below 5 million/ml in 3 consecutive samples, the subjects were required to discontinue all other forms of contraception for one year, during which the weekly injections of testosterone were continued. 18 of the subjects became azoospermic within 20 weeks of testosterone treatment, the other 15 remained severely oligozoospermic with a mean sperm density of 2.0 ± 0.8 million/ml at that time. Multiple blood sampling over the week following the first injection and after 16 weeks of testosterone treatment demonstrated that there were no differences in plasma concentrations of total or bioavailable testosterone or of oestradiol between the two groups of men. Similarly, the rate and degree of suppression of gonadotrophin secretion was similar in the two groups.

A brief history of the discovery of the anaesthetic potential of nitrous oxide is given followed by a more detailed history of the discovery of its specific toxic depressant effect on bone marrow. The work demonstrating that this effect is due to nitrous oxide reacting with vitamin B12 and, consequently, inhibiting methionine synthase is described. As the clinical results of this enzymatic inhibition in inhibiting DNA synthesis are mediated through both the folate and methionine metabolic pathways, these are discussed. Various theories as to how this mediation occurs are contrasted. The clinical relevance of these potential toxic side effects are discussed. Methods of assessing the toxicity of nitrous oxide are analysed and it is concluded that they are either too insensitive or require highly invasive sampling. The use of the formiminoglutamic acid urine excretion test in assessing folate metabolism is appraised and the possibility of using this test to assess nitrous oxide toxicity evaluated. Raised excretion is an index of folate metabolic abnormalities. This thesis then examines the toxicity of nitrous oxide in clinical practice by using this test. Finally, the place of nitrous oxide in modern anaesthesia is discussed.