The endocannabinoid system functions through two well characterized receptor systems termed CB₁ and CB₂.  However a growing body of evidence exists that suggest that the system is more complicated and indicate the presence of additional receptors.  Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological activities. In this thesis it is demonstrated that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GPR55 with nM potencies.  Ligands having no CB₁ or CB₂ activity that are believed to function at some of these novel receptor types such as cannabidiol and abnormal cannabidiol effect activity of GPR55.  These data suggest that GRP55 is a novel cannabinoid receptor, and its relative preference for ligands with respect to CB₁and CB₂ described here will permit delineation of its physiological function.  Data also suggesting that GRP55 couples to Gα13 and can mediate activation of RhoA, rac1 and cdc42.  It’s also demonstrated that GPR55 is functionally expressed in the mouse brain and using CB₁ and GPR55 selective ligands it is possible to functionally distinguish these receptors. It is therefore probable that both these receptors contribute to cannabinoid activity in CNS function.

The modulation of L-type calcium current (ICa) and the catecholamine-induced chloride current (ICl,cAMP) by G-protein coupled regulatory pathways were studied in isolated guinea pig cardiac ventricular myocytes using the whole cell patch clamp and flash photolysis techniques. A number of novel findings are reported in this thesis. The rapid release of GTP, the natural ligand of G-proteins, from its inert caged precursor produced a large enhancement of ICa which could be detected within 20 ms of the photolysing light pulse. A fast component of this response persisted under conditions of current rundown and inhibition of cAMP-dependent phosphorylation. This suggests the involvement of a membrane-delimited pathway not dependent on soluble second messengers. The photorelease of the non-hydrolysable GTPγS caused a biphasic increase in ICa in the majority of myocytes and a sustained response in the others. Pipette dialysis with GTPγS had a three-fold effect: pertussis toxin-sensitive inhibition of the ICa responses to isoprenaline, forskolin and photoreleased GTP; competitive inhibition of the enhancement of ICa by further photoreleased GTPγS; and modulation of the kinetics of cAMP-dependent activation of ICl,cAMP and ICa without any significant changes in their magnitudes. The flash photolysis of caged cAMP produced large increases in both ICl,cAMP and ICa but the latter was more than twice as sensitive to cAMP (EC50 = ~ 1.1 μM and ~ 0.47 μM). Urotensin has recently been identified as the ligand for a previously orphaned G-protein coupled receptor, and has been shown to be a potent chronic vasoconstrictor. This thesis reports an additional modulatory effect on ICl,cAMP. μM urotensin had no effect on its own but potentiated responses to sub-maximal sympathetic stimulation. Estrogen reduces forskolin- and isoprenaline-stimulated cAMP accumulation in the rat heart and inhibits cardiac ICa via a G-protein. However, the application of μM ß-estradiol to guinea pig myocytes in the presence of low doses of either forskolin (0.5 μM) or isoprenaline (20 nM) produced large increases in ICl,cAMP. This effect was mediated by a cell surface receptor. The involvement of nitric oxide synthase (NOS) was not required, unlike in acute estrogenic responses in vascular endothelia. Raloxifene, a selective estrogen receptor modulator (SERM), was similarly able to potentiate the results of sympathetic stimulation but with a much slower time course.

No description available.

No description available.

No description available.

No description available.

This research primarily focused on identifying the formulation parameters which control the efficacy of liposomes as delivery systems to enhance the delivery of poorly soluble drugs. Preliminary studies focused on the drug loading of ibuprofen within vesicle systems. Initially both liposomal and niosomal formulations were screened for their drug-loading capacity: liposomal systems were shown to offer significantly higher ibuprofen loading and thereafter lipid based systems were further investigated. Given the key role cholesterol is known to play within the stability of bilayer vesicles. the optimum cholesterol content in terms of drug loading and release of poorly soluble drugs was then investigated. From these studies a concentration of 11 total molar % of cholesterol was used as a benchmark for all further formulations. Investigating the effect of liposomc composition on several low solubility drugs, drug loading was shown to be enhanced by adopting longer chain length lipids. cationic lipids and. decreasing drug molecular weight. Drug release was increased by using cationic lipids and lower molecular weight of drug; conversely, a reduction was noted when employing longer chain lipids thus supporting the rational of longer chain lipids producing more stable liposomes, a theory also supported by results obtained via Langmuir studies· although it was revealed that stability is also dependent on geometric features associated with the lipid chain moiety. Interestingly, reduction in drug loading appeared to be induced when symmetrical phospholipids were substituted for lipids constituting asymmetrical alkyl chain groups thus further highlighting the importance of lipid geometry. Combining a symmetrical lipid with an asymmetrical derivative enhanced encapsulation of a hydrophobic drug while reducing that of another suggesting the importance of drug characteristics. Phosphatidylcholine liposornes could successfully be prepared (and visualised using transmission electron microscopy) from fatty alcohols therefore offering an alternative liposomal stabiliser to cholesterol. Results obtained revealed that liposomes containing tetradecanol within their formulation shares similar vesicle size, drug encapsulation, surface charge. and toxicity profiles as liposomes formulated with cholesterol, however the tetradecanol preparation appeared to release considerably more drug during stability studies. Langmuir monolayer studies revealed that the condensing influence by tetradecanol is less than compared with cholesterol suggesting that this reduced intercalation by the former could explain why the tetradecanol formulation released more drug compared with cholesterol formulations. Environmental scanning electron microscopy (ESEM) was used to analyse the morphology and stability of liposomes. These investigations indicated that the presence of drugs within the liposomal bilayer were able to enhance the stability of the bilayers against collapse under reduced hydration conditions. In addition the presence of charged lipids within the formulation under reduced hydration conditions compared with its neutral counterpart. However the applicability of using ESEM as a new method to investigate liposome stability appears less valid than first hoped since the results are often open to varied interpretation and do not provide a robust set of data to support conclusions in some cases.

The decomposition of drugs in the solid state has been studied using aspirin and salsalate as models. The feasibility of using suspension systems for predicting the stability of these drugs in the solid state has been investigated. It has been found that such systems are inappropriate in defining the effect of excipients on 'the decomposition of the active drug due to chqnges in the degradation pathway. Using a high performance liquid chromatographic method, magnesium stearate was shown to induce the formation of potentlally immunogenic products in aspirin powders. These products which included salicylsalicylic acid .and acetylsalicyclsalicylic acid were not detected in aspirin suspensions which had undergone the same extent of decomposition. By studying the effect of pH and of added excipients on the rate of decomposition of aspirin in suspension systems, it has been shown that excipients such as magnesium stearate containing magnesium oxide, most probably enhance the decomposition of both aspirin and salsalate by alkalinising the aqueous phase. In the solid state, pH effects produced by excipients appear to be relatively unimportant. Evidence is presented to suggest that the critical parameter is a depression in melting point induced by: the added excipient. Microscopical examination in fact showed the formation of clear liquid layers in aspirin samples containing added magnesium stearate but not in control samples. Kinetic equations which take into account both the diffusive barrier presented by the liquid films and the. geometry of the aspirin crystals were developed. Fitting of the .experimental data to these equations showed good agreement. with the postulated theory. Monitorjng of weight issues during the decomposition of aspirin revealed that in the solid systems studied where the bulk of the decomposition product sublimes, it is possible to estimate the extent of degradation from the residual weight, provided the initial weight is known. The corollary is that in such open systems, monitoring of decomposition products is inadequate for assessing the extent of decomposition. In addition to the magnesium stearate-aspirin system, mapyramine maleate-aspirin mixtures were used to model interactive systems. Work carried out in an attempt to stabilise such systems included microencapsulation and film coating. The protection obtained was dependent on the interactive species used. Gelatin for example appeared to stabilise aspirin against the adverse effects of magnesium stearate but increased its decomposition in the presence of mapyramine maleate.

Disturbances of cation homeostasis, particularly hypomagnesaemia, are a frequent consequence of treatment with aminoglycoside antibiotics. These disturbances are thought to result from renal wasting of cations and administration of gentamicin to rats has been shown to produce hypercalciuria and hypermagnesiuria. The aims of this study were to attempt to elucidate these responses in anaesthetised rats infused with gentamicin and to use this model to investigate the mechanisms of these effects. Fischer 344 rats were anaesthetised and surgically prepared for clearance experiments. Infusion of gentamicin in isotonic saline increased urinary output of calcium and magnesium while sodium and potassium output were unaffected. These elevations in calcium and magnesium excretion were explained by reduced tubular reabsorption of these cations. Both the hypercalciuric and hypermagnesiuric responses to gentamicin were extremely rapid and were sustained during drug infusion; when gentamicin infusion ceased both responses were rapidly reversible. Infusion of another aminoglycoside, tobramycin, produced very similar effects to gentamicin. The hypercalciuria and hypermagnesiuria caused by gentimicin infusion were unaffected by parathyroidectomy. The peak increases in calcium and magnesium output brought about by infusion of gentamicin with frusemide were not significantly different to the increases produced by frusemide alone. The site at which gentamicin interferes with calcium and magnesium reabsorption cannot be firmly deduced from these results. However, the known close association between calcium and sodium reabsorption in the proximal tubule implies that gentamicin is unlikely to change proximal calcium reabsorption without a similar change in proximal sodium reabsorption. The similarity between the hypercalciuric and hypermagnesiuric effects of frusemide alone and the effects of frusemide infused simultaneously with gentamicin suggests that gentamicin may act at the same site as the diuretic, the thick ascending limb of the loop of Henle.

No description available.