No description available.

Deuterium labeled methadone and deuterium labeled metabolites were synthesized to use in gas chromatography mass spectrometry (GCMS) studies of the metabolic pathways of metha-done in rats. These compounds were also useful to develop sensitive and selective analytical methods to study the pharmacokinetics and disposition of methadone. Synthesis of the deuterium labeled compounds was mainly achieved by using known procedures with special treatments required to provide label enrichment. Using the labeled and unlabeled derivatives, mass fragmentation processes that are common to methadone and its metabolites were defined. Aryl ring migration was observed in a fragmentation process for 2-ethylidene-l,5-dimethyl-3,3-diphenyl pyrrolidine (EDDP). This aryl ring migration was not a favorable process for ring substituted EDDP analogs. Various aspects regarding the optimization of the selected ion monitoring (SIM) analysis of methadone and its metabolites in biological fluids are described. The SIM analysis using deuterium labeled compounds as internal standards generally proved to be selective but not as sensitive as expected using electron impact ionization (EI) conditions of GCMS. One advantage of using SIM over GC analysis was described in terms of ratio analysis. Quantitation of methadone in human plasma and saliva using SIM gave a lower limit of sensitivity of 20 ng/0.5 ml of sample by monitoring the base peak, m/e 72. The mean methadone ratios of saliva to plasma for two patients were 0.55 ± 0.15 (standard deviation) and 0.48 ± 0.10 (standard deviation). Methadone metabolism studies emphasized the detection of minor metabolites using special extraction methods for rat bile and using labeled and unlabeled compounds. Comparison of the mass spectra from total ion current (TIC) profiles of metabolites from unlabeled compounds with those from labeled compounds run as separate experiments gave GCMS evidence for methadone nitrone (N-methylene-l-methyl-3,3-diphenyl-4-oxo-hexanamine-oxide). Possibilities for the metabolic formation of N-hydroxynormethadone and the pharmacological significance of the detection of methadone nitrone were described. A proposal for metabolic studies to examine the potential formation of other methadone metabolites resulting from metabolic oxidation of nitrogen was presented. Structural evidence for the methadone nitrone molecule was obtained indirectly by chemical oxidation studies of metha done metabolites. m-Chloroperbenzoic acid treatment of EDDP perchlorate gave three products: methadone nitrone, 4,4-diphen yl-2,5-heptanedione (diketone), and 2-acetyl-5-methyl-3,3-di-phenyl-l-pyrroline. These compounds were identified from their IR, NMR and mass spectral data. Mass fragmentation processes were defined for the methadone nitrone. Possible mechanisms for the formation of methadone nitrone and diketone from chemical oxidation of EDDP are proposed. Since diazepam is a drug widely abused by methadone maintenance patients, methadone-diazepam interaction studies were designed to analyze metabolites using deuterium labeled authentic compounds as internal standards. Metabolites in the conjugated fraction of rat bile were analyzed using deuterium labeled biosynthetic internal standards. Diazepam (5 mg/kg) was given to rats through a cannulated jugular vein and a subcutaneous dose of methadone (10 mg/kg) was given. Bile was collected through the cannulated bile duct over a period of 24 hours. The deuterium label was found to be stable even under severe conditions of incubation temperature and time. SIM analysis of bile sample extracts showed that concomitant administration of diazepam with methadone did not affect biliary excretion of EDDP nor the conjugated metabolites. This indicates that diazepam does not interact with methadone at the hepatic metabolism level and with transport of the metabo-: lites by the biliary excretion route. Application of the use of a biosynthetic internal standard to drug metabolism and pharmacokinetic studies by means of ratio analysis was described with examples. / Pharmaceutical Sciences, Faculty of / Graduate

High Performance Liquid Chromatography is currently used for the purpose of analytical evaluation of drugs. This is mainly because it allows the separation method both, a qualitative and a quantitative, analysis of high selectivity mixture evaluation and sensitivity. The diploma thesis deals with the issue of purity evaluation of pharmaceutical substance Nalbufin hydrochloride. The aim of the experimental part of the diploma thesis deals with the development and validation of the analytical method for assessing the purity of Nalbufin hydrochloride.An HPLC method was developed on a Nova-Pak C18 column. The mobile phase consisted of two components A and B. MF A composition was as follows: 0.97 g of sodium octane sulfonate was dissolved in 900 mL of water to which were added 100 mL of acetonitrile and 2 mL of triethylamine. Created solution was treated with phosphoric acid to pH 2.5. MF B had the following composition: 0.86g of sodium octanesulfonate was dissolved in 800 mL of water to which 200 mL was added acetonitrile of 2 mL of TEA. The resulting pH was adjusted to pH 2.5 with phosphoric acid. gradient MF had the following composition: From zero minutes from 100% A to 30 min. to 0% A. The 30-60min. 0% A, 60-61 min. with a linear change to 100% A, 61-70 min. equilibrium into the original conditions to 100% A at a flow rate of 1 mL/min. In the following section we evaluated the basic validation parameters: linear dynamic range 0.3 - 4.5 g/mL, we calculated the linear regression equation for Nalbuphine in R2 (0.9999), Oxycodone R2 (0.9999) and Noroxycodone R2 (0.9998). The method gave us detection limits for Nalbuphine 0.069 g/mL. Oxycodone had a detection limit of 0.053 g/mL and Noroxycodone 0.048mg / mL. The limist of quantification in these cases were 0.209 g / mL for Nalbuphine, 0.161 g/mL for Oxycodone and 0.147 g/mL for Noroxycodone. Repeatability for the limit of quantification was also set expressed by the relative standard deviation. For Nalbuphine - RSD = 0.40%, Oxycodone - RSD = 2.39% and Noroxycodone - RSD = 1.25% (RSD 7.0%). The following validation parameter was accuracy. The resulting RSD was 0.44% (RSD 5.0%). The last evaluated parameter was robustness. For pH 2.4, the change value was resolution of 1.5% and repeatability of RSD = 0.85%. The change of resolution value for pH 2.6 was 2.8% and repeatability RSD = 1.29% (max. 5% limit). The second factor observed for the robustness was the temperature change of the column. The arithmetic average was calculated from the individual peak areas and relative standard deviation RSD = 3.45% was evaluated with the change of resolution, which had a value of 3.34%. Thus, we can state that the developed chromatographic method has been verified in the givenvalidation parameters and is suitable for determining the purity of Nalbuphine hydrochloride.

Local anaesthetics have been implemented extensively in the case of a variety of painful superficial procedures, venipuncture, skin graft harvesting, anal or genital pruritus, poison ivy rashes, postherpetic neuralgia and several other dermatoses. The dilemma with commercially available local acting anaesthetics is that it may take well up to an hour to produce an anaesthetic effect. Anaesthetics have to traverse the highly efficient barrier, the stratum corneum, in order to reach the intended target site which is the free nerve endings located in the dermis. The objective of this study was to compare the transdermal delivery of an eutectic combination of two ionisable amide types of local anaesthetics, lidocaine HCI and prilocaine HCI, delivered with the novel Pheroid™ technology to that of a commercially available product in order to establish whether the lag time could be significantly reduced. Several techniques of promoting the penetration of these anaesthetics have previously been employed, including occlusive dressing, entrapment in liposomes and miscelles, iontophoretic delivery and so forth. The Pheroid™ delivery system is novel technology that entails improved delivery of several active compounds. It is a submicron emulsion type formulation that possesses the ability to be transformed in morphology and size, thereby affording it tremendous flexibility. Since it primarily consists of unsaturated essential fatty acids, it is not seen as foreign to the body but rather as a skin-friendly carrier. Vertical Franz cell diffusion studies were performed over a 12 hour period using Caucasian female abdominal skin obtained, with the consent of the donor, from abdominoplastic surgery. Comparison was made between the commercial product EMLA® cream, the active local anaesthetics dissolved in phosphate buffered solution (PBS) and the active ingredients entrapped within Pheroid™ vesicles. Distinct entrapment could be ascertained visually by confocal laser scanning microscopy (CLSM). The amount of drug that traversed the epidermal membrane into the receptor phase was then assayed by high performance liquid chromatography (HPLC). The results obtained with the Pheroid™ vesicles revealed a biphasic character with rapid permeation during the first two hours, followed by a plateau between 3 to 12 hours. The initial dramatic increase in percentage yield and flux indicates that the Pheroid™ carrier enhances the transdermal delivery of the actives in order to accelerate the onset of action. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

Local anaesthetics have been implemented extensively in the case of a variety of painful superficial procedures, venipuncture, skin graft harvesting, anal or genital pruritus, poison ivy rashes, postherpetic neuralgia and several other dermatoses. The dilemma with commercially available local acting anaesthetics is that it may take well up to an hour to produce an anaesthetic effect. Anaesthetics have to traverse the highly efficient barrier, the stratum corneum, in order to reach the intended target site which is the free nerve endings located in the dermis. The objective of this study was to compare the transdermal delivery of an eutectic combination of two ionisable amide types of local anaesthetics, lidocaine HCI and prilocaine HCI, delivered with the novel Pheroid™ technology to that of a commercially available product in order to establish whether the lag time could be significantly reduced. Several techniques of promoting the penetration of these anaesthetics have previously been employed, including occlusive dressing, entrapment in liposomes and miscelles, iontophoretic delivery and so forth. The Pheroid™ delivery system is novel technology that entails improved delivery of several active compounds. It is a submicron emulsion type formulation that possesses the ability to be transformed in morphology and size, thereby affording it tremendous flexibility. Since it primarily consists of unsaturated essential fatty acids, it is not seen as foreign to the body but rather as a skin-friendly carrier. Vertical Franz cell diffusion studies were performed over a 12 hour period using Caucasian female abdominal skin obtained, with the consent of the donor, from abdominoplastic surgery. Comparison was made between the commercial product EMLA® cream, the active local anaesthetics dissolved in phosphate buffered solution (PBS) and the active ingredients entrapped within Pheroid™ vesicles. Distinct entrapment could be ascertained visually by confocal laser scanning microscopy (CLSM). The amount of drug that traversed the epidermal membrane into the receptor phase was then assayed by high performance liquid chromatography (HPLC). The results obtained with the Pheroid™ vesicles revealed a biphasic character with rapid permeation during the first two hours, followed by a plateau between 3 to 12 hours. The initial dramatic increase in percentage yield and flux indicates that the Pheroid™ carrier enhances the transdermal delivery of the actives in order to accelerate the onset of action. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

by Yiu-kuen Kam. / Bibliography: leaves 108-118 / Thesis (M.Phil.) -- Chinese University of Hong Kong, 1983

No description available.

Integrins are transmembrane heterodimer protein receptors that mediate adherence to both the intracellular cytoskeleton and extracellular matrix. They play a major role in cellular adhesion and the breadth of their importance in biology is only recently being understood. The ability to detect concentrations of integrins on the cell surface, spatially resolve them, and study the dynamics of their behavior would be a significant advance in this field. Ultimately, the ability to detect dynamic changes of integrins on the surface of a cell maybe possible by developing a combined device such as an atomic force microscope (AFM) and surface enhanced Raman spectroscopy (SERS) system. However, the focus of this research is to first determine if integrins can be detected using SERS. Surface enhanced Raman spectroscopy (SERS) is technique used to detect the presence of analytes at the nanomolar level or below, through detection of inelastically scattered light. This thesis discusses the detection of integrins employing SERS as the detection modality. Integrins have been detected, in solution, using two silver colloids as the enhancing surface. Two silver colloid preparation methods are compared by ease of formulation and degree of enhancement in this thesis. Citrate and hydroxylamine hydrochloride (HA-HCl) reduced silver colloids were prepared through wet chemistry,compared using UV-Vis light spectroscopy, and tested for surface enhancement using adenine (a strong SERS active molecule), and two different integrins, (alpha)V(beta)3 and (alpha)5(beta)1. Results indicated that both colloids demonstrate SERS activity for varying concentrations of adenine as compared to standard non-enhanced Raman, however, only the citrate reduced colloid showed significant enhancement effect for the integrins.

Integrins are transmembrane heterodimer protein receptors that mediate adherence to both the intracellular cytoskeleton and extracellular matrix. They play a major role in cellular adhesion and the breadth of their importance in biology is only recently being understood. The ability to detect concentrations of integrins on the cell surface, spatially resolve them, and study the dynamics of their behavior would be a significant advance in this field. Ultimately, the ability to detect dynamic changes of integrins on the surface of a cell maybe possible by developing a combined device such as an atomic force microscope (AFM) and surface enhanced Raman spectroscopy (SERS) system. However, the focus of this research is to first determine if integrins can be detected using SERS. Surface enhanced Raman spectroscopy (SERS) is technique used to detect the presence of analytes at the nanomolar level or below, through detection of inelastically scattered light. This thesis discusses the detection of integrins employing SERS as the detection modality. Integrins have been detected, in solution, using two silver colloids as the enhancing surface. Two silver colloid preparation methods are compared by ease of formulation and degree of enhancement in this thesis. Citrate and hydroxylamine hydrochloride (HA-HCl) reduced silver colloids were prepared through wet chemistry,compared using UV-Vis light spectroscopy, and tested for surface enhancement using adenine (a strong SERS active molecule), and two different integrins, (alpha)V(beta)3 and (alpha)5(beta)1. Results indicated that both colloids demonstrate SERS activity for varying concentrations of adenine as compared to standard non-enhanced Raman, however, only the citrate reduced colloid showed significant enhancement effect for the integrins.

Staphylococcus aureus protease V8 (SPV8), also known as Endoproteinase Glu-C (EC 3.4.21.19), is an enzyme isolated from the bacteria Staphylococcus aureus. This unusual enzyme has been found to cleave specifically at glutamyl and aspartyl peptide bonds and has been used as a tool in the preparation of protein substrates for amino acid sequence analysis. SPV8 has been reported to show some stability toward various denaturants (Drapeau, G.R. (1977) Methods in Enzymology_, 47:189-191). In order to more adequately assess the denaturant stability of SPV8, the effect of guanidine hydrochloride (HC1), a common protein denaturant, on the proteolytic action of SPV8 was studied. The extent of cleavage of the glutamyl peptide bond in adrenocorticotropic hormone 1-10 (ACTH 1-10) was found to decrease with increasing concentrations of guanidine HC1.At 22°C in the presence of 3.0 M guanidine HCl, only 25% of SPV8's proteolytic activity was retained. In the presence of 4.0, 5.0, or 6.0 M guanidine HC1, virtually all proteolytic activity toward the glutamyl bond of ACTH 1-10 was lost, presumably due to the inactivation of the protease by denaturation or increased autolysis mediated by the guanidine HC1. At temperatures above 22°C, SPV8 was more susceptible to inactivation by guanidine HC1. Thus SPV8 appears to retain some proteolytic activity in the presence of guanidine HC1, but only at concentrations less than 4.0 M. There was no difference in the proteolytic activity of SPV8 toward the glutamyl peptide bond of ACTH 1-10 when incubation was carried out in ammonium bicarbonate buffer (pH 7.80), phosphate buffer (pH 7.80), or Tris-HC1 buffer (pH 7.80). The presence of 1 mM calcium chloride in the 3.0 M guanidine HC1/phosphate buffer solution enhanced the enzymatic action of SPV8. The presence of 1 mM calcium chloride in Tris-HC1 buffer (pH 7.80) does not effect the proteolytic activity of SPV8 at 22°C. However, there was slight reduction in SPV8's enzymatic action toward ACTH 1-10 when the 1 mM calcium chloride was present in the 3.0 M guanidine HC1/ammonium bicarbonate buffer (pH 7.80) solution. / Department of Chemistry