The neurotoxic effects of enzyme inhibitors at the neuromuscular junction

Current knowledge of the long-term, low dose effects of carbamate (CB) anti-cholinesterases on skeletal muscle or on the metabolism and regulation of the molecular forms of acetylcholinesterase (AChE) is limited. This is largely due to the reversible nature of these inhibitors and the subtle effects they induce which has generally made their study difficult and preliminary investigations were conducted to determine suitable study methods. A sequential extraction technique was used to rapidly analyse AChE molecular form activity at the mouse neuromuscular junction and also in peripheral parts of muscle fibres. AChE in the synaptic cleft involved in the termination of cholinergic transmission was successfully assessed by the assay method and by an alternative method using a correlation equation which represented the relationship between synaptic AChE and the prolongation of extra-cellular miniature endplate potentials. It was found that inhibition after in vivo Carbamate (CB) dosing could not be maintained during tissue analysis because CB-inhibited enzyme complexes decarbamoylated vary rapidly and could not be prevented even when maintained on ice. The methods employed did not therefore give a measure of inhibition but presented a profile of metabolic responses to continual, low dose CB treatment. Repetitive and continual infusion with low doses of the CBs: pyridostigmine and physostigmine induced a variety of effects on mouse skeletal muscle. Both compounds induced a mild myopathy in the mouse diaphragm during continual infusion which was characterised by endplate deformation without necrosis; such deformation persisted on termination of treatment but had recovered slightly 14 days later. Endplate and non-endplate AChE molecular forms displayed selective responses to CB treatment. During treatment endplate AChE was reduced whereas non-endplate AChE was largely unaffected, and after treatment, endplate AChE recovered, whereas non-endplate AChE was up-regulated. The mechanisms by which these responses become manifest are unclear but may be due to CB-induced effects on nerve-mediated muscle activity, neurotrophic factors or morphological and physiological changes which arise at the neuromuscular junction. It was concluded that, as well as inhibiting AChE, CBs also influence the metabolism and regulation of the enzyme and induce persistent endplate deformation; possible detrimental effects of long-term, low-dose determination requires further investigation.

Event related potential (ERP) evidence for selective impairment of verbal recollection in abstinent recreational methylenedioxymethamphetamine (“Ecstasy”)/polydrug users

Objectives Ecstasy is a recreational drug whose active ingredient, 3,4-methylenedioxymethamphetamine (MDMA), acts predominantly on the serotonergic system. Although MDMA is known to be neurotoxic in animals, the long-term effects of recreational Ecstasy use in humans remain controversial but one commonly reported consequence is mild cognitive impairment particularly affecting verbal episodic memory. Although event-related potentials (ERPs) have made significant contributions to our understanding of human memory processes, until now they have not been applied to study the long-term effects of Ecstasy. The aim of this study was to examine the effects of past Ecstasy use on recognition memory for both verbal and non-verbal stimuli using ERPs. Methods We compared the ERPs of 15 Ecstasy/polydrug users with those of 14 cannabis users and 13 non-illicit drug users as controls. Results Despite equivalent memory performance, Ecstasy/polydrug users showed an attenuated late positivity over left parietal scalp sites, a component associated with the specific memory process of recollection. Conlusions This effect was only found in the word recognition task which is consistent with evidence that left hemisphere cognitive functions are disproportionately affected by Ecstasy, probably because the serotonergic system is laterally asymmetrical. Experimentally, decreasing central serotonergic activity through acute tryptophan depletion also selectively impairs recollection, and this too suggests the importance of the serotonergic system. Overall, our results suggest that Ecstasy users, who also use a wide range of other drugs, show a durable abnormality in a specific ERP component thought to be associated with recollection.

Studies on the neurotoxicity of 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) in SH-SY5Y cells

We have studied the hypothesis that 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) is neurotoxic. Salsolinol induced a significant time and dose related inhibition of 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazoyl blue (MTT) reduction, and increased lactate dehydrogenase release (LDH) release from human SH-SY5Y neuroblastoma cells, at concentrations within the range of 1-methyl-4-phenylpyridinium (MPP+) cytotoxicity, in vitro. Cytotoxicity was not inhibited by the addition of antioxidants, monoamine oxidase inhibitors or imipramine. In confluent monolayers, salsolinol stimulated catecholamine uptake with EC50 values of 17 muM and 11 muM, for noradrenaline and dopamine, respectively. Conversely, at concentrations above 100 muM, salsolinol inhibited the uptake of noradrenaline and dopamine, with IC50 values of 411 muM and 379 muM, respectively. The inhibition of catecholamine uptake corresponded to the increase displacement of [3H]nisoxetine from the uptake 1 site by salsolinol, as the Ki (353 muM) for displacement was similar to the IC50 (411 and 379 muM) for uptake. Salsolinol stimulated catecholamine uptake does not involve the uptake recognition site, or elevation of cAMP, cGMP, or inhibition of protein kinase C. Salsolinol also inhibited both carbachol (1 mM) and K+ (100 mM, Na+ adjusted) evoked released of noradrenaline from SH-SY5Y cells, with IC50 values of 500 muM and 120 muM, respectively. In conclusion, salsolinol appears to be cytotoxic to SH-SY5Y cells, via a mechanism that does not require uptake 1, bioactivation by monoamine oxidase, or membrane based free radical damage. The effects of salsolinol on catecholamine uptake, and the mechanism of toxicity require further investigation.

Aluminium administered in drinking water but not in the diet influences biopterin metabolism in the rodent

To investigate the neurotoxic effects of aluminium (Al) Al was administered: 1) in the diet of the rat (30 mg Al/kg body weight for 6 weeks); 2) as a suspension of aluminium acetate in drinking water of the rat for 3 months and 3) in a long-term study in the mouse in which aluminosilicates were incorporated into a pelleted diet (1035 mg/kg of food over 23 months). In the latter treatment, increased Al was combined with a reduction in calcium and magnesium; a treatment designed to increase absorption of Al into the body. Administration of Al in the drinking water significantly reduced total brain biopterins and BH4 synthesis. However, no significant affect of Al in the diet on total biopterins or BH4 synthesis was found either in the rat or in the long-term study in the mouse. In addition, in the mouse no significant effects of the Al diet on levels of noradrenaline, serotonin, dopamine, 5-HIAA or CAT could be demonstrated. Hence, the occurrence of brain alterations may depend on the Al species present and the method of administration. Al salts in drinking water may increase brain tissue levels compared with the administration of a more insoluble species. Since alterations in biopterin metabolism are also a feature of Alzheimer's disease (AD) these results support the hypothesis that Al in the water supply may be a factor in AD.

Genotypic and phenotypic properties of coagulase-negative staphylococci causing dialysis catheter-related sepsis

Sixty coagulase-negative staphylococcus (CNS) isolates were recovered from the blood cultures or peritoneal dialysate effluent of 43 patients on renal dialysis. The patients had either renal dialysis catheter-related sepsis (CRS) or continuous ambulatory peritoneal dialysis (CAPD)-associated peritonitis. Isolates were characterized by biotyping, and genotyped by pulsed-field gel electrophoresis (PFGE). Phenotypic properties of the strains were also investigated. Several genotypes were identified with no one specific strain of CNS being associated with CRS. However, closely related strains were isolated from several patients within the units studied, suggesting horizontal transfer of micro-organisms. Genotypic macro-restriction profiles did not concur with phenotypic profiles or biotypes, confirming that genotyping is required for epidemiological studies. All staphylococcal strains were investigated for the production of phenotypic characteristics. Significant differences were predominantly seen in the production of lipase, esterase and elastase in strains isolated from the renal patients with CRS and CAPD-associated peritonitis, compared with a non-septic control group. These phenotypic characteristics may therefore have a role in the maintenance of CRS in renal patients. © 2003 The Hospital Infection Society. Published by Elsevier Science Ltd. All rights reserved.

The influence of aluminium, administered in the diet and in drinking water, on biopterin metabolism

To investigate the neurotoxic effects of aluminium (Al) three studies were carried out in which Al was administered: 1) in the diet, 2) as a suspension of aluminium acetate in drinking water and 3) a long-term study in which aluminosilicates were incorporated into a pelleted diet. Admistration of Al in the drinking water significantly reduced total brain biopterin. However, no significant affect of Al in the diet on total bipterins or BH4 synthesis was found.

Single-cell ELISA and flow cytometry as methods for highlighting potential neuronal and astrocytic toxicant specificity

The timeline imposed by recent worldwide chemical legislation is not amenable to conventional in vivo toxicity testing, requiring the development of rapid, economical in vitro screening strategies which have acceptable predictive capacities. When acquiring regulatory neurotoxicity data, distinction on whether a toxic agent affects neurons and/or astrocytes is essential. This study evaluated neurofilament (NF) and glial fibrillary acidic protein (GFAP) directed single-cell (S-C) ELISA and flow cytometry as methods for distinguishing cell-specific cytoskeletal responses, using the established human NT2 neuronal/astrocytic (NT2.N/A) co-culture model and a range of neurotoxic (acrylamide, atropine, caffeine, chloroquine, nicotine) and non-neurotoxic (chloramphenicol, rifampicin, verapamil) test chemicals. NF and GFAP directed flow cytometry was able to identify several of the test chemicals as being specifically neurotoxic (chloroquine, nicotine) or astrocytoxic (atropine, chloramphenicol) via quantification of cell death in the NT2.N/A model at cytotoxic concentrations using the resazurin cytotoxicity assay. Those neurotoxicants with low associated cytotoxicity are the most significant in terms of potential hazard to the human nervous system. The NF and GFAP directed S-C ELISA data predominantly demonstrated the known neurotoxicants only to affect the neuronal and/or astrocytic cytoskeleton in the NT2.N/A cell model at concentrations below those affecting cell viability. This report concluded that NF and GFAP directed S-C ELISA and flow cytometric methods may prove to be valuable additions to an in vitro screening strategy for differentiating cytotoxicity from specific neuronal and/or astrocytic toxicity. Further work using the NT2.N/A model and a broader array of toxicants is appropriate in order to confirm the applicability of these methods.

A novel and rational approach towards designing aluminium chelators

Aluminium (Al) is known to be neurotoxic and has been associated with the aetiology of Alzheimer's Disease. To date, only desferrioxamine (DFO), a trihydroxamic acid siderophore has been used in the clinical environment for the removal of Al from the body. However, this drug is expensive, orally inactive and is associated with many side effects. These studies employed a theoretical approach, with the use of quantum mechanics (QM) via semi-empirical molecular orbital (MO) calculations, and a practical approach using U87-MG glioblastoma cells as a model for evaluating the influence of potential chelators on the passage of aluminium into cells. Preliminary studies involving the Cambridge Structural Database (CSD) identified that Al prefers binding to bidentate ligands in a 3:1 manner, whereby oxygen was the exclusive donating atom. Statistically significant differences in M-O bond lengths when compared to other trivalent metal ions such as Fe3+ were established and used as an acceptance criterion for subsequent MO calculations. Of the semi-empirical methods parameterised for Al, the PM3 Hamiltonian was found to give the most reliable final optimised geometries of simple 3:1 Al complexes. Consequently the PM3 Hamiltonian was used for evaluating the Hf of 3:1 complexes with more complicated ligands. No correlation exists between published stability constants and individual parameters calculated via PM3 optimisations, although investigation of the dicarboxylates reveals a correlation of 0.961 showing promise for affinity prediction of closely related ligands. A simple and inexpensive morin spectrofluorescence assay has been developed and optimised producing results comparable to atomic absorption spectroscopy methods for the quantitative analysis of Al. This assay was used in subsequent in vitro models, initially on E. coli, which indicated that Al inhibits the antimicrobial action of ciprofloxacin, a potent quinolone antibiotic. Ensuing studies using the second model, U87-MG cells, investigated the influence of chelators on the transmembrane transport of Al, identifying 1,2-diethylhydroxypyridin-4-one as a ligand showing greatest potential for chelating Al in the clinical situation. In conclusion, these studies have explored semi-empirical MO Hamiltonians and an in-vitro U87-MG cell line, both as possible methods for predicting effective chelators of Al.

The percutaneous absorption of ionisable compounds

The effects of ionisation on transdermal drug delivery using excised human epidermis (HS) and silastic rubber (SR) as model permeation barriers were investigated in vitro using Franz-type absorption cells. Suspensions and solutions of salicylic acid (SA), the model ionogenic permeant, were used as donors and the variables studied were vehicle pH and trans-membrane pH-gradients. For solutions, the pH effect was related to the level of ionisation of the drug and the degree of saturation of the solution. With suspensions, the observed permeation rate was unaffected by pH. The penetration profiles through HS and SR were similar, although the overall flux through HS was about 70% of that observed through SR. Pretreatment of the membranes with various enhancer regimens, including oleic acid, Azone and N, N-dimethylamides in propylene glycol (PG) and isopropyl myristate (IPM) promoted the penetration of SA. SR was not a suitable model for enhancer pretreatment using IPM as a vehicle as the membrane was significantly disrupted by this vehicle. The results from comparable experiments with and without a trans-membrane pH-gradient did not have a significant effect upon flux or flux enhancement after pretreatment with the above enhancers. A theoretical model for the extraction coefficients of weak acids was derived using the partition coefficients of the ionised and unionised species, pH and pKa. This model was shown to account for the variation in overall partition of salicylic acid dependent upon pH and pKa. This model was shown to account for the variation in overall partition of salicylic acid dependent upon pH and pKa. The distribution of this solute between aqueous and oily phases, with and without added enhancer, was measured as a function of pH. The extraction coefficients determined were consistent with the model and showed that the behaviour of the system can be explained without referral to ion-pair mechanisms. Phosphonoacetate is an effective antiviral agent. However, as it is charged at physiological pH, its permeation across cell membranes is limited. To assess the improvement of the transport properties of this molecule, mono-, di- and tri-ester prodrugs were examined. These were assessed for stability and subsequent breakdown with respect to pH by HPLC. In vitro percutaneous absorption was observed using the triester, but not the ionic mono- or di-esters. The triester absorption could be potentiated using a range of enhancers with oleic acid being the most effective. Cyclodextrins (CD) have a role as absorption enhancers for peptide compounds across nasal epithelium. One potential mode of action is that CDs include these compounds, protect them from enzymic attack and thereby increase their residence time in the nasal epithelium. This study investigated the potential of CDs to protect ester prodrugs from enzymatic breakdown and prevent production of poorly transportable ionic species. Using a range of CD to ester molar ratios (10:1 to 2500:1) a small, but measurable, protection for the model esters (parabens) against esterase attack was observed. Possible mechanisms for this phenomenon are that CDs include the ester, making it unavailable for hydrolysis, the CDs may also affect the esterase in some way preventing access for the ester into the active site.

Gram-positive infections in patients with end-stage renal disease

Gram-positive microorganisms, specifically coagulase-negative staphylococci are the most common species recovered from clinical culture specimens of patients with end-stage renal disease. The propensity of coagulase-negative staphylococci (CNS) to cause infection in this patient group has been widely debated. However, it is still unclear how this usually avirulent commensal microorganism produces infection that contributes to high rates of morbidity and mortality in patients with end-stage renal disease. The aim of this thesis was to investigate the rate, geographical distribution, molecular and phenotypic mechanisms of Gram-positive microorganisms associated with infection in renal dialysis patients. In addition, it sought to assess the value of early serological diagnosis of dialysis catheter-associated infection and the effect of antimicrobial treatment regimens on the faecal carriage of enteric microorganisms. In this study, the incidence of haemodialysis catheter-associated infection was established with the Meditrend audit tool. This tool was used to assess the infection outcomes of catheter insertion and management procedures until the catheter was explanted. Introduction of a catheter management protocol decreased the incidence of catheter-related infection. Staphylococcal species recovered from episodes of haemodialysis catheter-associated infection and continuous ambulatory peritoneal dialysis (CAPD)-associated peritonitis were genotyped by determination of macrorestriction profiles with pulsed-field gel electrophoresis. This highlighted horizontal transfer of microorganisms between different patients and the environment. The phenotypic characteristics of these strains were also investigated to determine characteristics that could be used as markers for dialysis catheter-associated infection. The expression of elastase, lipase and esterase by CNS was significantly associated with infection. A rapid enzyme-linked immunosorbent assay incorporating a novel staphylococcal antigen (lipid S) was used to evaluate the early detection of anti-staphylococcal immunoglobulin gamma in patient sera. The comparison of culture positive and culture negative patients demonstrated a steady state of immune activation in both groups. However anti-lipid S serum antibody titres > 1000 were found to be a predictor of infection. The effect on faecal carriage of vancomycin resistant enterococci (VRE) and Clostridium difficile toxins in patients treated with CAPD when empiric cephalosporin therapy was substituted for piperacillin/tazobactam was investigated. The introduction of piperacillin/tazobactam demonstrated a decrease in the faecal carriage of VRE.

Development of a post-mitotic, neuronal-astrocytic cell system, for the prediction of acute neurotoxicity in humans

The human NT2.D1 cell line was differentiated to form both a 1:2 co-culture of post-mitotic NT2 neuronal and NT2 astrocytic (NT2.N/A) cells and a pure NT2.N culture. The respective sensitivities to several test chemicals of the NT2.N/A, the NT2.N, and the NT2.D1 cells were evaluated and compared with the CCF-STTG1 astrocytoma cell line, using a combination of basal cytotoxicity and biochemical endpoints. Using the MTT assay, the basal cytotoxicity data estimated the comparative toxicities of the test chemicals (chronic neurotoxin 2,5-hexanedione, cytotoxins 2,3- and 3,4-hexanedione and acute neurotoxins tributyltin- and trimethyltin- chloride) and also provided the non-cytotoxic concentration-range for each compound. Biochemical endpoints examined over the non-cytotoxic range included assays for ATP levels, oxidative status (H2O2 and GSH levels) and caspase-3 levels as an indicator of apoptosis. although the endpoints did not demonstrate the known neurotoxicants to be consistently more toxic to the cell systems with the greatest number of neuronal properties, the NT2 astrocytes appeared to contribute positively to NT2 neuronal health following exposure to all the test chemicals. The NT2.N/A co-culture generally maintained superior ATP and GSH levels and reduced H2O2 levels in comparison with the NT2.N mono-culture. In addition, the pure NT2.N culture showed a significantly lower level of caspase-3 activation compared with the co-culture, suggesting NT2 astrocytes may be important in modulating the mode of cell death following toxic insult. Overall, these studies provide evidence that an in vitro integrated population of post-mitotic human neurons and astrocytes may offer significant relevance to the human in vivo heterogeneous nervous system, when initially screening compounds for acute neurotoxic potential.

Characterisation of coagulase-negative staphylococci associated with endocarditis

Coagulase-negative staphylococci are major aetiological agents of prosthetic valve endocarditis and an occasional cause of native valve disease. It is currently unclear how this group of usually avirulent microorganisms produces an infection associated with high rates of morbidity and mortality. The aim of this thesis was to investigate whether there are specific genotypes and/or phenotypes of coagulase-negative staphylococci with a propensity to cause infective endocarditis and to investigate any identified virulence factors as markers of infection. In this study, strains of endocarditis-related coagulase-negative staphylococci were genotyped by determining their macrorestriction genomic profile using pulsed-field gel electrophoresis. The strains were also investigated for phenotypic characteristics that predisposed the microorganisms to infect heart valves. By comparing coagulase-negative staphylococcal strains recovered from endocarditis patients with isolates from other significant infections (prosthetic device-related osteomyelitis and catheter-associated sepsis), no specific genotype or phenotype with a predilection to cause endocarditis was identified. However, the majority of the endocarditis-associated and other infection strains expressed the potential virulence factors lipase and esterase. Another approach to the investigation of virulence determinants used patient's serum to screen a Staphylococcus epidermidis NCTC 11047 genomic DNA library for cellular and secreted staphylococcal products that were expressed in vivo. The characterisation of two clones, which reacted with serum collected from a S. epidermidis-related endocarditis patient identified a staphylococcal pyruvate dehydrogenase complex E2 subunit and a novel secreted protein with homology to a Staphylococcus aureus staphyloxanthin biosynthesis protein and a secreted protein of unknown function described in Staphylococcus carnosus. Investigation of the secreted protein previously undetected in S. epidermidis, termed staphylococcal secretory antigen (SsaA), identified a potential marker of S. epidermidis-related endocarditis.

Down-regulation of central glucocorticoid receptor expression using antisense oligonucleotide technology

Endogenous glucocorticoids and serotonin have been implicated in the pathophysiology of depression, anxiety and schizophrenia. This thesis investigates the potential of downregulating expression of central Type II glucocorticoid receptors (GR) both in vitro and in vivo, with empirically-designed antisense oligodeoxynucleotides (ODN), to characterise GR modulation of 5-HT2A receptor expression using quantitative RT-PCR, Western blot analysis and radioligand binding. The functional consequence of GR downregulation is also determined by measuring 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane hydrochloride (DOI) mediated 5-HT2A receptor specific headshakes. Using a library of random antisense ODN probes, RNAse H accessibility mapping of T7-primed, in vitro transcribed GR mRNA revealed several potential cleavage sites and identified an optimally effect GR antisense ODN sequence of 21-mer length (GRAS5). In vitro efficacy studies using rat C6 glioma cells showed a 56% downregulation in GR mRNA levels and 80% downregulation in GR protein levels. In the same cells a 29% upregulation in 5-HT2A mRNA levels and 32% upregulation in 5-HT2A protein levels was revealed. This confirmed the optimal nature of the GRAS5 sequence to produce marked inhibition of GR gene expression, and also revealed GR modulation of the 50-HT2A receptor subtype in C6 glioma cells to be a tonic repression of receptor expression. The distribution of a fluorescently-labelled GRAS5 ODN was detected in diverse areas of the rat brain after single ICV administration, although this fluorescence signal was not sustained over a period of 5 days. However, fluorescently-labelled GRAS5 ODN, when formulated in polymer microspheres, showed diverse distribution in the brain which was maintained for 5 days following a single ICV administration. This produced no apparent neurotoxic effects on rat behaviour and hypothalamic-pituitary-adrenal (HPA) axis homeostasis. Furthermore, a single polymer microsphere injection ICV proved to be an effective means of delivering antisense ODNs and this was adopted for the in vivo efficacy studies. In vivo characterisation of GRAS5 revealed marked downregulation of GR mRNA in rat brain regions such as the frontal cortex (26%), hippocampus (35%), and hypothalamus (39%). Downregulation of GR protein was also revealed in frontal cortex (67%), hippocampus (76%), and hypothalamus (80%). In the same animals upregulation of 5-HT2A mRNA levels was shown in frontal cortex (13%), hippocampus (7%), and hypothalamus (5%) while upregulation in 5-HT2A protein levels was shown in frontal cortex (21 %). This upregulation in 5-HT2A receptor density as a result of antisense-mediated inhibition of GR was further confirmed by a 55% increase in DOl-mediated 5-HT2A receptor specific headshakes. These results demonstrate that GR is involved in tonic inhibitory regulation of 5-HT2A receptor expression and function in vivo, thus providing the potential to control 5-HT2A-linked disorders through corticosteroid manipulation. These experiments have therefore established an antisense approach which can be used to investigate pharmacological characteristics of receptors.

Prodrugs of antiviral phosphonates

Phosphonoformate and phosphonoacetate are effective antiviral agents, however they are charged at physiological pH and as such penetration into cells and diffusion across the blood-brain bamer is limited. In an attempt to increase the lipophilicity and improve the transport properties of these molecules, prodrugs were synthesised and their stabilities and reconversion to the parent compound subsequently investigated by the techniques of 31P nuclear magnetic resonance spectroscopy and high performance liquid Chromatography. A series of 4-substituted dibenzyl (methoxycarbonyl)phosphonates were prepared and found to be hydrolytically unstable giving predominantly the diesters, benzyl (methoxycarbonyl)phosphonates. This instability arose from the electron-withdrawing effect of the carbonyl group promoting nucleophilic attack at phosphorus. It was possible to influence the mechanism and, to some extent, the rate of hydrolysis of the phosphonoformate triesters to the diesters by varying the electronic nature of the substituent in the 4-position of the aromatic ring. Strongly electron-withdrawing groups increased the sensitivity of phosphorus to nucleophilic attack, thus promoting P-O .bond cleavage and rapid hydrolysis. Conversely, weakly electron-withdrawing substituents encouraged C-O bond fission, presumably through resonance stabilisation of the benzyl carbonium ion. The loss of the protecting group on phosphorus was in competition with nucleophilic attack at the carbonyl group, resulting in P-C bond cleavage with dibenzyl phosphite formation. The high instability and P-C bond fission make triesters unsuitable prodrug forms of phosphonoformate. A range of chemically stable triesters of phosphonoacetate were synthesised and their bioactivation investigated. Di(benzoyloxymethyl) (methoxycarbonylmethyl)phosphonates degraded to the relevant benzoyloxymethyl (methoxycarbonylmethyl)phosphonate in the presence of esterase. The enzymatic activation was restricted to the removal of only one protecting group from phosphorus, most likely due to the close proximity of the benzoyloxy ester function to the anionic charge on the diester. However, in similar systems di(4-alkanoyloxybenzyl) (methoxycarbonylmethyl)phosphonates degraded in the presence of esterase with the loss of both protecting groups on phosphorus to give the monoester, (methoxycarbonylmethyl)phosphonate, via the intermediary of the unstable 4-hydroxy benzyl esters. The methoxycarbonyl function remained intact. The rate of enzymatic hydrolysis and subsequent removal of the protecting groups on phosphorus was dependent on the nature of the alkanoyl group and was most rapid for the 4-nbutanoyloxybenzyl and 4-iso-butanoyloxybenzyl esters of phosphonoacetate. This provides a strategy for the design of a prodrug with sufficient stability in plasma to reach the central nervous system in high concentration, wherein rapid metabolism to the active drug by brain-associated enzymes occurs.

Reduced metal transferrin binding in neurological diseases

By employing G75 gel-filtration chromotography, it has been demonstrated that human plasma gallium speciation (and by implication, Al speciation) is bimodal. Normally, gallium was predominantly bound to a high molecular weight fraction which was presumably transferrin. Literature reviews and experimental work throughout this thesis provided evidence to support this idea. An aluminium-transferrin species was assumed to be relatively non-toxic and a protective function for this complex has been suggested. A second, low molecular weight species of gallium was observed and its identity has been suggested to be citrate. The results of this thesis support the concept citrate was a gallium binding ligand present in the plasma, but there was another species (tentatively identified as phosphate) which bound gallium to a much greater degree than did citrate in the majority of samples studied. The consequence of a low molecular weight species of aluminium is the possibility that this leads to a more rapid, uncontrolled deposition of the metal in the brain compared to a transferrin mediated mechanism. Plasma speciation studies in Alzheimer's disease, Parkinson's disease, Down's syndrome, and neonates has revealed an altered ratio of the two gallium species found in control subjects. In all groups there was an increase in the potentially more neurotoxic low molecular weight species. These observations have led to a suggested mechanism of accumulation of metals in the brain, which is known to occur in the first three groups. Possible pathogenic mechanisms are described. The results can also offer an explanation to the reported increased sensitivity to the toxic effects of aluminium in the neonate. Speciation studies on normal plasma has shown the balance between high and low molecular weight species of gallium to be influenced by many physiological factors. There appears to be a fine equilibrium between both species which can be altered without any great difficulty. Therefore, in the diseased groups studied, it is possible that there are subtle biochemical changes within the circulatory system to affect the equilibrium which results in an increased low molecular weight species of aluminium. Furthermore, it has been demonstrated that there is a group of normal controls with no clinical signs of Alzheimer's or Parkinson's disease which have reduced transferrin binding. This indicates there is a population of healthy people who are at risk to the development of either disease.