Structural studies on the capsular antigens of some Escherichia coli serotypes
- Authors: Leslie, Margaret Ruth
- Date: 1995
- Subjects: Escherichia Polysaccharides Antigens Enterobacteriaceae
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3767 , http://hdl.handle.net/10962/d1003245
- Description: The research presented in this thesis forms part of an on-going collaborative programme concerned with the determination of the chemical structures of the surface antigens of bacteria belonging to genera within the family Enterobacteriaceae. Bacteria of this family are opportunistic pathogens and are frequently responsible for serious infections in animals and man. Surface antigens produced by virulent strains are largely polysaccharides and occur as lipopolysaccharides (the O-antigens) and capsular polysaccharides (the K-antigens) respectively. The extracellular polysaccharide antigens expressed by strains of the species Escherichia coli are of considerable . interest due to their effect on immunological processes and the relationship which exists between their chemical structure and virulence. To date, some seventy-four K-antigens have been distinguished serologically within the species E. coli and structures have been determined for most of these. The K-antigens of E. coli are structurally diverse and exhibit serological cross-reactivity with other pathogenic bacteria. The structures of five previously unstudied E. coli K-antigens, viz. those produced by serotypes 020:K1 01 :H-, 08:K45:H9, 08:K50:H-, 0101 :K1 03:H-, and 08:K43:H11, are presented in this thesis. A variety of chemical techniques has been employed in the structural analysis, and these are discussed. Two-dimensional NMR spectroscopic techniques proved invaluable for the structural elucidation of these complex carbohydrates, and high-field NMR spectroscopy alone was used in the analysis of the K43 antigen. Structural analysis of the K1 03 antigen was facilitated by specific enzymatic degradation, using a bacteriophage-borne endoglycanase. The K45 antigen was found to contain the unusual sugar 3-acetamido-3,6-dideoxygalactopyranose, while the K50 and K103 antigens join a minority group of polysaccharides which contain pyruvate as their only acidic component.
- Full Text:
- Authors: Leslie, Margaret Ruth
- Date: 1995
- Subjects: Escherichia Polysaccharides Antigens Enterobacteriaceae
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3767 , http://hdl.handle.net/10962/d1003245
- Description: The research presented in this thesis forms part of an on-going collaborative programme concerned with the determination of the chemical structures of the surface antigens of bacteria belonging to genera within the family Enterobacteriaceae. Bacteria of this family are opportunistic pathogens and are frequently responsible for serious infections in animals and man. Surface antigens produced by virulent strains are largely polysaccharides and occur as lipopolysaccharides (the O-antigens) and capsular polysaccharides (the K-antigens) respectively. The extracellular polysaccharide antigens expressed by strains of the species Escherichia coli are of considerable . interest due to their effect on immunological processes and the relationship which exists between their chemical structure and virulence. To date, some seventy-four K-antigens have been distinguished serologically within the species E. coli and structures have been determined for most of these. The K-antigens of E. coli are structurally diverse and exhibit serological cross-reactivity with other pathogenic bacteria. The structures of five previously unstudied E. coli K-antigens, viz. those produced by serotypes 020:K1 01 :H-, 08:K45:H9, 08:K50:H-, 0101 :K1 03:H-, and 08:K43:H11, are presented in this thesis. A variety of chemical techniques has been employed in the structural analysis, and these are discussed. Two-dimensional NMR spectroscopic techniques proved invaluable for the structural elucidation of these complex carbohydrates, and high-field NMR spectroscopy alone was used in the analysis of the K43 antigen. Structural analysis of the K1 03 antigen was facilitated by specific enzymatic degradation, using a bacteriophage-borne endoglycanase. The K45 antigen was found to contain the unusual sugar 3-acetamido-3,6-dideoxygalactopyranose, while the K50 and K103 antigens join a minority group of polysaccharides which contain pyruvate as their only acidic component.
- Full Text:
Structural studies on some enterobacterial capsular antigens
- Authors: Whittaker, Darryl Vanstone
- Date: 1994
- Subjects: Bacterial antigens -- Analysis Antigens Enterobacteriaceae Escherichia coli Klebsiella
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3803 , http://hdl.handle.net/10962/d1003281
- Description: The investigations presented in this thesis form part of a systematic international effort to establish the structures of the capsules produced by the bacterial genera, Escherichia coli and Klebsiella (family enterobacteriaceae). These bacteria are of medical interest as they are opportunistic pathogens and are frequently responsible for serious infections in animals and man. Invasive strains are invariably surrounded by a structurally complex polysaccharide capsule which contributes to the organism's ability to attenuate non-specific host defence mechanisms or, in some instances, to completely prevent an immune response. A knowledge of the chemical composition and structure of the capsule is, therefore, of great value as it provides insight into the mechanisms involved in this process. The E. coli, in particular, have generated considerable interest as their capsules are more structurally diverse and cross-reactivity with other, more pathogenic bacteria has also been demonstrated. Accordingly, the structures of three previously unstudied E. coli K-antigens viz. those produced by serotypes 020:K83:H26, 020:K84:H26, and 09:K48:H9 have been established by chemical and spectroscopic means and are presented in this thesis. In addition, a reinvestigation of the structure of the capsule produced by Klebsiella K15 using a novel enzymatic approach was also undertaken and a revised structure is proposed . The E. coli K48 polysaccharide is of special interest as it was found to contain a new diacetamido trideoxy hexose hitherto unrecorded. A synthesis for this saccharide is also presented. Finally, the application of lithium dissolved in ethylenediamine for the degradation of amino sugar-containing polysaccharides was also investigated using the capsular polysaccharides produced by E. coli serotypes K38 and K84 as model compounds.
- Full Text:
- Authors: Whittaker, Darryl Vanstone
- Date: 1994
- Subjects: Bacterial antigens -- Analysis Antigens Enterobacteriaceae Escherichia coli Klebsiella
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3803 , http://hdl.handle.net/10962/d1003281
- Description: The investigations presented in this thesis form part of a systematic international effort to establish the structures of the capsules produced by the bacterial genera, Escherichia coli and Klebsiella (family enterobacteriaceae). These bacteria are of medical interest as they are opportunistic pathogens and are frequently responsible for serious infections in animals and man. Invasive strains are invariably surrounded by a structurally complex polysaccharide capsule which contributes to the organism's ability to attenuate non-specific host defence mechanisms or, in some instances, to completely prevent an immune response. A knowledge of the chemical composition and structure of the capsule is, therefore, of great value as it provides insight into the mechanisms involved in this process. The E. coli, in particular, have generated considerable interest as their capsules are more structurally diverse and cross-reactivity with other, more pathogenic bacteria has also been demonstrated. Accordingly, the structures of three previously unstudied E. coli K-antigens viz. those produced by serotypes 020:K83:H26, 020:K84:H26, and 09:K48:H9 have been established by chemical and spectroscopic means and are presented in this thesis. In addition, a reinvestigation of the structure of the capsule produced by Klebsiella K15 using a novel enzymatic approach was also undertaken and a revised structure is proposed . The E. coli K48 polysaccharide is of special interest as it was found to contain a new diacetamido trideoxy hexose hitherto unrecorded. A synthesis for this saccharide is also presented. Finally, the application of lithium dissolved in ethylenediamine for the degradation of amino sugar-containing polysaccharides was also investigated using the capsular polysaccharides produced by E. coli serotypes K38 and K84 as model compounds.
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A structural study of the capsular antigen of Klebsiella serotype K43
- Authors: Aereboe, Michael
- Date: 1993
- Subjects: Polysaccharides , Klebsiella , Antigens , Enterobacteriaceae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3740 , http://hdl.handle.net/10962/d1003218 , Polysaccharides , Klebsiella , Antigens , Enterobacteriaceae
- Description: This thesis presents a detailed chemical and spectroscopic determination of the capsular, polysaccharide K-antigen isolated from the Klebsiella bacterium, serotype K43 (culture #2482). The repeating unit of the capsular polysaccharide was found to be of the "3 + 2" repeating unit type. A uronic acid was found as part of a disaccharide side chain and the main chain of the polysaccharide was found to be composed of a neutral trisaccharide of mannose and galactose. The work forms part of an ongoing research interest in bacterial polysaccharides of this laboratory and now completes the structural elucidation of all the Klebsiella K-antigens, bar three antigens which were originally assigned to other laboratories. These data together with the respective serological characteristics of each serotype are available to the molecular biologist, and may result in the production of: vaccine(s) against Klebsiella infections, diagnostic products and novel carrier molecules enabling targeted drug delivery.
- Full Text:
- Authors: Aereboe, Michael
- Date: 1993
- Subjects: Polysaccharides , Klebsiella , Antigens , Enterobacteriaceae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3740 , http://hdl.handle.net/10962/d1003218 , Polysaccharides , Klebsiella , Antigens , Enterobacteriaceae
- Description: This thesis presents a detailed chemical and spectroscopic determination of the capsular, polysaccharide K-antigen isolated from the Klebsiella bacterium, serotype K43 (culture #2482). The repeating unit of the capsular polysaccharide was found to be of the "3 + 2" repeating unit type. A uronic acid was found as part of a disaccharide side chain and the main chain of the polysaccharide was found to be composed of a neutral trisaccharide of mannose and galactose. The work forms part of an ongoing research interest in bacterial polysaccharides of this laboratory and now completes the structural elucidation of all the Klebsiella K-antigens, bar three antigens which were originally assigned to other laboratories. These data together with the respective serological characteristics of each serotype are available to the molecular biologist, and may result in the production of: vaccine(s) against Klebsiella infections, diagnostic products and novel carrier molecules enabling targeted drug delivery.
- Full Text:
Pharmacodynamics of phenylpropanolamine: aspects of safety and efficacy in humans
- Authors: Petrie, Lauri René
- Date: 1993
- Subjects: Phenylpropanolamine
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3785 , http://hdl.handle.net/10962/d1003263 , Phenylpropanolamine
- Description: Phenylpropanolamine (PPA), a synthetic sympathomimetic amine, is widely used as a nasal decongestant and as an appetite suppressant. Much controversy exists regarding the efficacy of the drug as an anorectic agent, the related adverse reactions caused by the relatively high doses required for appetite suppression and the potential of this drug for abuse. Whilst numerous studies have been carried out to assess the central and cardiovascular safety of PPA and many investigations have been performed to evaluate efficacy in terms of weight loss in humans, there is a relative paucity of information regarding the effects of PPA on appetite and food intake. A pilot trial was conducted to determine the feasiblility of a multidimensional approach to evaluate the safety and efficacy of PPA as ananorectic agent in humans. Eight normotensive caucasian women who were overweight participated in a randomised double-blind cross-over comparison of PPA (75 mg) and placebo and were dosed to steady-state on a 12-hour fixed-dose schedule for a period of eleven weeks. Aspects of efficacy evaluated included the effects of PPA on hunger, appetite and satiety,salivation, macro-nutrient food intake and body weight. Standardised scales were used to quantitatively assess the possible subjective mood and behavioural reinforcing effects of PPA. Supine systolic and diastolic blood pressures were monitored continually throughout the trial. In addition, peak and trough blood samp1es were taken to monitor serum concentrations of PPA reached at steady-state and patient compliance with the dosing schedule. An adaptation of a published reverse-phase high performance liquid chromatographic (HPLC) assay for PPA in serum using U.V. detection at 210 nm is presented. A significant decrease in body weight, salivation, total food intake and carbohydrate consumption was demonstrated following PPA administration. Phenylpropanolamine produced significant decrements in subjective reports of hunger and appetite, whilst apparently having little effect on satiety. No significant changes were observed for blood pressures and PPA did not produce significant mood alterations or behavioural reinforcing effects. The study demonstrates the feasibility of using this muti-faceted approach, with certain design modifications, to evaluate the overall safety and efficacy of PPA as an appetite suppressant.
- Full Text:
- Authors: Petrie, Lauri René
- Date: 1993
- Subjects: Phenylpropanolamine
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3785 , http://hdl.handle.net/10962/d1003263 , Phenylpropanolamine
- Description: Phenylpropanolamine (PPA), a synthetic sympathomimetic amine, is widely used as a nasal decongestant and as an appetite suppressant. Much controversy exists regarding the efficacy of the drug as an anorectic agent, the related adverse reactions caused by the relatively high doses required for appetite suppression and the potential of this drug for abuse. Whilst numerous studies have been carried out to assess the central and cardiovascular safety of PPA and many investigations have been performed to evaluate efficacy in terms of weight loss in humans, there is a relative paucity of information regarding the effects of PPA on appetite and food intake. A pilot trial was conducted to determine the feasiblility of a multidimensional approach to evaluate the safety and efficacy of PPA as ananorectic agent in humans. Eight normotensive caucasian women who were overweight participated in a randomised double-blind cross-over comparison of PPA (75 mg) and placebo and were dosed to steady-state on a 12-hour fixed-dose schedule for a period of eleven weeks. Aspects of efficacy evaluated included the effects of PPA on hunger, appetite and satiety,salivation, macro-nutrient food intake and body weight. Standardised scales were used to quantitatively assess the possible subjective mood and behavioural reinforcing effects of PPA. Supine systolic and diastolic blood pressures were monitored continually throughout the trial. In addition, peak and trough blood samp1es were taken to monitor serum concentrations of PPA reached at steady-state and patient compliance with the dosing schedule. An adaptation of a published reverse-phase high performance liquid chromatographic (HPLC) assay for PPA in serum using U.V. detection at 210 nm is presented. A significant decrease in body weight, salivation, total food intake and carbohydrate consumption was demonstrated following PPA administration. Phenylpropanolamine produced significant decrements in subjective reports of hunger and appetite, whilst apparently having little effect on satiety. No significant changes were observed for blood pressures and PPA did not produce significant mood alterations or behavioural reinforcing effects. The study demonstrates the feasibility of using this muti-faceted approach, with certain design modifications, to evaluate the overall safety and efficacy of PPA as an appetite suppressant.
- Full Text:
A study of the biopharmaceutics and pharmacokinetics of the macrolide antibiotic, erythromycin
- Authors: Terespolsky, Susan Ann
- Date: 1992
- Subjects: Erythromycin -- Bioavailability , Erythromycin -- Pharmacokinetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3795 , http://hdl.handle.net/10962/d1003273 , Erythromycin -- Bioavailability , Erythromycin -- Pharmacokinetics
- Description: Erythromycin, a macrolide antibiotic isolated from Streptomyces erythreus, was first introduced into clinical medicine in 1952. It is active against most gram-positive bacteria, some gram-negative bacteria and is currently the agent of choice for Legionella pneumophila. Erythromycin is an acid-labile compound rapidly degrading in acidic solutions such as the acid environment of the stomach. As such, erythromycin absorption following oral administration of solid dosage forms is relatively poor. Accordingly there have been various approaches used to protect the drug against gastric inactivation. These precautions include enteric-coating of tablets, capsules or pellets of erythromycin base, the synthesis of acid stable 2' esters of erythromycin (ethylsuccinate and propionate) and salts of these esters (erythromycin estolate), and more recently, the synthesis of a range of new acid-stable, semi-synthetic macrolide antibiotics. The 2' esters are antimicrobially inactive or much less active than the parent compound and must be converted to the free erythromycin base in vivo in order to exhibit antibacterial activity. Intrinsic dissolution rates determined on raw material can provide extremely useful information relating to the gastrointestinal absorption of drugs from solid dosage forms. The large inter- and intrasubject variability associated with erythromycin base has, to date, mainly been attributed to gastric acid inactivation of the drug. However, changes in duodenal pH resulting in altered solubility and intrinsic dissolution rates may account for the observed variability. Thus, the intrinsic dissolution rates of erythromycin base at pH 6.0, 6.5, 7.0, 7.5 and 8.0 were compared in order to investigate the possible effects of pH changes which may occur in the duodenal contents, on the in vivo dissolution and subsequent absorption of this compound. The standard intrinsic dissolution rate test procedure employing a rotating disc of pure erythromycin base powder which only allows for dissolution from a constant surface area, was adapted and the drug quantitatively determined by reversed phase high performance liquid chromatography (HPLC) using ultraviolet detection. Results of intrinsic dissolution studies at both 22°C and 37°C indicate that the solubility, and therefore the rate of dissolution of erythromycin base is pH dependent, being more soluble at pH 6.0 than pH 8.0 (an approximate 800 times and 1000 times reduction in the amount dissolved after 30 minutes, at 22°C and 37°C respectively, when the pH of the medium was increased from 6 to 8). Although the stability of erythromycin and its ester derivatives in aqueous acidic solutions has been well documented, very little has been reported on the compound's stability in organic solvents. Methanol is recommended by official drug compendia (U.S.P. and B.P.) for use in erythromycin identification tests as well as in the sample preparation steps during assay procedures. Thus, the effect of methanol and acetonitrile, organic solvents of similar polarities and densities, on the stability of erythromycin base, erythromycin ethylsuccinate, propionyl erythromycin and erythromycin estolate at room temperature (22°C ± 0.5°C), using HPLC with electrochemical detection, was investigated. Erythromycin base is relatively stable in both methanol and acetonitrile, remaining intact for over 168 hours in acetonitrile and showing less than 5% degradation in methanol over the same period. Erythromycin ethylsuccinate in acetonitrile shows less than 5% degradation over 168 hours whereas in methanol, rapid hydrolysis occurs resulting in almost total conversion to base within 40 hours. Approximately 87% of erythromycin propionyl ester remained intact after 168 hours in acetonitrile whilst methanol caused rapid hydrolysis to erythromycin base (35% remaining after 28 hours). Erythromycin estolate appeared to be unstable in both acetonitrile and methanol. In acetonitrile, only 13% of the estolate remained intact after 168 hours, whereas in methanol, the reaction was much more rapid with 35% of the estolate remaining after 28 hours. The use of methanol as a solvent for erythromycin estolate reference standards is thus contraindicated. A number of conflicting reports on the half- life as well as the body compartment model that best describes erythromycin base serum concentration-time profiles (lBCM generally used to describe orally administered erythromycin, whilst a 2BCM has been used to describe erythromycin administered intravenously), appear in the literature. These differences may be largely attributed to the sampling period (between 6 and 12 hours) used in the repective studies. The objective of this study was to determine the body compartment model that best describes erythromycin base serum concentration-time curves by increasing the sampling time to 24 hours. In addition, the effect of chronic dosing of erythromycin on erythromycin pharmacokinetics, in the same group of subjects, was investigated. The single and multiple oral dose pharmacokinetics of erythromycin enteric coated base pellets within a gelatin capsule (250mg), were studied in 6 healthy, normal volunteers (19.5 ± 0.76 years, 71.5 ± 8.18 kg, 180.33 ± 5.99 cm). Furthermore, steady state concentrations were predicted using the pharmacokinetic parameters obtained from the single dose study, and compared with those obtained in the multiple dose study. Plasma concentrations were determined using a sensitive high-performance liquid chromatographic method with electrochemical detection. For the single dose study, after a tlag of 2.5 ± 0.71 hr, Cmax (1.12 ± 0.47 μ/ml) was reached at a tmax of 4.08 ± 0.93 hr post dose, with serum concentrations ranging from 0.31 - 1.62 μ/ml. The half-life was found to be 5.42 ± 1.31 hr. On multiple dosing (250mg six hourly), serum concentrations for the fifth, ninth and thirteenth dosing intervals ranged from 0.67 - 2.92 μ/ml, 1.69 - 3.65 μ/ml and 0.61 - 3.01 μ/ml, occurring at 3.75 ± 0.69 hr, 3.17 ± 1.03 hr and 3.17 ± 1.03 hr post dose with a Cmax of 1.89 ± 0.68 μ/ml, 2.35 ± 0.70 μ/ml and 1.94 ± 0.74 μ/ml, respectively. The area under the serum concentration- time curve for the single dose study (AUC₀₋∞) was 4.67 ± 0.88 hr.μ/ml, whilst the AUC₀₋τ. for the fifth, ninth and thirteenth dosing intervals of the multiple dose study were 5.77 ± 1.76 hr.μ/ml, 6.46 ± 1.33 hr.μ/ml and 5.97 ± 2.36 hr.μ/ml respectively, indicating an approximately 33% increase in AUC on chronic dosing of erythromycin. The observed increase in AUC may be a result of increased bioavailability or a decrease in clearance on chronic dosing.
- Full Text: false
- Authors: Terespolsky, Susan Ann
- Date: 1992
- Subjects: Erythromycin -- Bioavailability , Erythromycin -- Pharmacokinetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3795 , http://hdl.handle.net/10962/d1003273 , Erythromycin -- Bioavailability , Erythromycin -- Pharmacokinetics
- Description: Erythromycin, a macrolide antibiotic isolated from Streptomyces erythreus, was first introduced into clinical medicine in 1952. It is active against most gram-positive bacteria, some gram-negative bacteria and is currently the agent of choice for Legionella pneumophila. Erythromycin is an acid-labile compound rapidly degrading in acidic solutions such as the acid environment of the stomach. As such, erythromycin absorption following oral administration of solid dosage forms is relatively poor. Accordingly there have been various approaches used to protect the drug against gastric inactivation. These precautions include enteric-coating of tablets, capsules or pellets of erythromycin base, the synthesis of acid stable 2' esters of erythromycin (ethylsuccinate and propionate) and salts of these esters (erythromycin estolate), and more recently, the synthesis of a range of new acid-stable, semi-synthetic macrolide antibiotics. The 2' esters are antimicrobially inactive or much less active than the parent compound and must be converted to the free erythromycin base in vivo in order to exhibit antibacterial activity. Intrinsic dissolution rates determined on raw material can provide extremely useful information relating to the gastrointestinal absorption of drugs from solid dosage forms. The large inter- and intrasubject variability associated with erythromycin base has, to date, mainly been attributed to gastric acid inactivation of the drug. However, changes in duodenal pH resulting in altered solubility and intrinsic dissolution rates may account for the observed variability. Thus, the intrinsic dissolution rates of erythromycin base at pH 6.0, 6.5, 7.0, 7.5 and 8.0 were compared in order to investigate the possible effects of pH changes which may occur in the duodenal contents, on the in vivo dissolution and subsequent absorption of this compound. The standard intrinsic dissolution rate test procedure employing a rotating disc of pure erythromycin base powder which only allows for dissolution from a constant surface area, was adapted and the drug quantitatively determined by reversed phase high performance liquid chromatography (HPLC) using ultraviolet detection. Results of intrinsic dissolution studies at both 22°C and 37°C indicate that the solubility, and therefore the rate of dissolution of erythromycin base is pH dependent, being more soluble at pH 6.0 than pH 8.0 (an approximate 800 times and 1000 times reduction in the amount dissolved after 30 minutes, at 22°C and 37°C respectively, when the pH of the medium was increased from 6 to 8). Although the stability of erythromycin and its ester derivatives in aqueous acidic solutions has been well documented, very little has been reported on the compound's stability in organic solvents. Methanol is recommended by official drug compendia (U.S.P. and B.P.) for use in erythromycin identification tests as well as in the sample preparation steps during assay procedures. Thus, the effect of methanol and acetonitrile, organic solvents of similar polarities and densities, on the stability of erythromycin base, erythromycin ethylsuccinate, propionyl erythromycin and erythromycin estolate at room temperature (22°C ± 0.5°C), using HPLC with electrochemical detection, was investigated. Erythromycin base is relatively stable in both methanol and acetonitrile, remaining intact for over 168 hours in acetonitrile and showing less than 5% degradation in methanol over the same period. Erythromycin ethylsuccinate in acetonitrile shows less than 5% degradation over 168 hours whereas in methanol, rapid hydrolysis occurs resulting in almost total conversion to base within 40 hours. Approximately 87% of erythromycin propionyl ester remained intact after 168 hours in acetonitrile whilst methanol caused rapid hydrolysis to erythromycin base (35% remaining after 28 hours). Erythromycin estolate appeared to be unstable in both acetonitrile and methanol. In acetonitrile, only 13% of the estolate remained intact after 168 hours, whereas in methanol, the reaction was much more rapid with 35% of the estolate remaining after 28 hours. The use of methanol as a solvent for erythromycin estolate reference standards is thus contraindicated. A number of conflicting reports on the half- life as well as the body compartment model that best describes erythromycin base serum concentration-time profiles (lBCM generally used to describe orally administered erythromycin, whilst a 2BCM has been used to describe erythromycin administered intravenously), appear in the literature. These differences may be largely attributed to the sampling period (between 6 and 12 hours) used in the repective studies. The objective of this study was to determine the body compartment model that best describes erythromycin base serum concentration-time curves by increasing the sampling time to 24 hours. In addition, the effect of chronic dosing of erythromycin on erythromycin pharmacokinetics, in the same group of subjects, was investigated. The single and multiple oral dose pharmacokinetics of erythromycin enteric coated base pellets within a gelatin capsule (250mg), were studied in 6 healthy, normal volunteers (19.5 ± 0.76 years, 71.5 ± 8.18 kg, 180.33 ± 5.99 cm). Furthermore, steady state concentrations were predicted using the pharmacokinetic parameters obtained from the single dose study, and compared with those obtained in the multiple dose study. Plasma concentrations were determined using a sensitive high-performance liquid chromatographic method with electrochemical detection. For the single dose study, after a tlag of 2.5 ± 0.71 hr, Cmax (1.12 ± 0.47 μ/ml) was reached at a tmax of 4.08 ± 0.93 hr post dose, with serum concentrations ranging from 0.31 - 1.62 μ/ml. The half-life was found to be 5.42 ± 1.31 hr. On multiple dosing (250mg six hourly), serum concentrations for the fifth, ninth and thirteenth dosing intervals ranged from 0.67 - 2.92 μ/ml, 1.69 - 3.65 μ/ml and 0.61 - 3.01 μ/ml, occurring at 3.75 ± 0.69 hr, 3.17 ± 1.03 hr and 3.17 ± 1.03 hr post dose with a Cmax of 1.89 ± 0.68 μ/ml, 2.35 ± 0.70 μ/ml and 1.94 ± 0.74 μ/ml, respectively. The area under the serum concentration- time curve for the single dose study (AUC₀₋∞) was 4.67 ± 0.88 hr.μ/ml, whilst the AUC₀₋τ. for the fifth, ninth and thirteenth dosing intervals of the multiple dose study were 5.77 ± 1.76 hr.μ/ml, 6.46 ± 1.33 hr.μ/ml and 5.97 ± 2.36 hr.μ/ml respectively, indicating an approximately 33% increase in AUC on chronic dosing of erythromycin. The observed increase in AUC may be a result of increased bioavailability or a decrease in clearance on chronic dosing.
- Full Text: false
A study of the effect of progesterone on the body weight regulation in intact female rats
- Authors: Ravelingien, Jo
- Date: 1992
- Subjects: Progesterone -- Physiological effect , Body weight -- Regulation , Rats -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3787 , http://hdl.handle.net/10962/d1003265 , Progesterone -- Physiological effect , Body weight -- Regulation , Rats -- Research
- Description: It is the aim of this study to elucidate the influence of progesterone on body weight regulation in intact female rats. A study of the literature includes a description of the body weight regulation and the effects of ovarian hormones on it. The controlled-system approach tries to link behavioral and physiological factors altering energy balance. The experimental study is subdivided into food-intake - and food-selection studies, a locomotor activity study, a study eliciting a possible role of thermogenesis, and finally rat liver studies which consist of a gas chromatography analysis of hepatic fatty acids and an electron microscopy study examining the ultrastructure of hepatocytes. It can be concluded that the effect of progesterone treatment on the body weight of intact female rats depends on the route of administration. There is a significant increase in body weight after subcutaneous progesterone injections without changes in total caloric intake and nutrient selection habits, indicating the importance of energy expenditure. But changes in spontaneous activity make no contribution in the progesterone-induced energy storage. It is also concluded that peripherally located brown adipose tissue thermogenesis is not changed, without ruling out the effect of more centrally located thermogenic organs as the liver. In this organ, small but significant changes in the fatty acid profile occur during the subcutaneous progesterone treatment.
- Full Text:
- Authors: Ravelingien, Jo
- Date: 1992
- Subjects: Progesterone -- Physiological effect , Body weight -- Regulation , Rats -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3787 , http://hdl.handle.net/10962/d1003265 , Progesterone -- Physiological effect , Body weight -- Regulation , Rats -- Research
- Description: It is the aim of this study to elucidate the influence of progesterone on body weight regulation in intact female rats. A study of the literature includes a description of the body weight regulation and the effects of ovarian hormones on it. The controlled-system approach tries to link behavioral and physiological factors altering energy balance. The experimental study is subdivided into food-intake - and food-selection studies, a locomotor activity study, a study eliciting a possible role of thermogenesis, and finally rat liver studies which consist of a gas chromatography analysis of hepatic fatty acids and an electron microscopy study examining the ultrastructure of hepatocytes. It can be concluded that the effect of progesterone treatment on the body weight of intact female rats depends on the route of administration. There is a significant increase in body weight after subcutaneous progesterone injections without changes in total caloric intake and nutrient selection habits, indicating the importance of energy expenditure. But changes in spontaneous activity make no contribution in the progesterone-induced energy storage. It is also concluded that peripherally located brown adipose tissue thermogenesis is not changed, without ruling out the effect of more centrally located thermogenic organs as the liver. In this organ, small but significant changes in the fatty acid profile occur during the subcutaneous progesterone treatment.
- Full Text:
Biopharmaceutics and pharmacokinetics of the macrolide antibiotic Josamycin
- Authors: Skinner, Michael Fredrick
- Date: 1992
- Subjects: Antibiotics -- Bioavailability , Antibiotics -- Pharmacokinetics
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3791 , http://hdl.handle.net/10962/d1003269
- Description: The investigations detailed herein have been conducted to address various aspects of the biopharmaceutics and pharmacokinetics of josamycin which to-date, have received little or no attention in the literature. Areas of investigation have included the selective determination of josamycin in serum and urine samples, the stability of josamycin in stored biological samples, intrinsic dissolution rates, solubility, acid and alkali stability and bioavailability and pharmacokinetics after dosing with a solution, powder and tablets. High performance liquid chromatography (HPLC) was used as the main analytical tool throughout these studies and proved to be highly versatile for the determination of josamycin in a number of different media. HPLC analysis afforded simple yet accurate determination of josamycin in samples from dissolution, solubility, tablet content and stability studies. Furthermore, the specificity afforded by HPLC was particularly useful for the separation of josamycin from degradation products formed in acid and alkali media. Since metabolites of josamycin are microbiologically active, microbiological assays do not determine the concentration solely of josamycin. An analytical method capable of the selective determination of josamycin in serum and urine samples is therefore required for the procurement of reliable bioavailability and pharmacokinetic data. HPLC affords this selectivity and a method for the selective determination of josamycin in serum and urine was successfully developed. The assay was simple yet precise, accurate and sensitive. Furthermore, it was well suited to the determination of josamycin in a large number of biological samples. Its success was largely due to the use of a solid phase extraction step using C₁₈ extraction columns, with a highly specific wash sequence followed by a phase separation step after elution from the extraction column. Chromatography was performed on a C₁₈ reversed-phase analytical column with UV detection of josamycin and internal standard at 231 nm and at 204 nm respectively using a programmable multi-wavelength detector. Only slight modification of the assay described should enable the selective determination of the metabolites of josamycin. This assay, therefore, lays the groundwork for future investigations into the pharmacokinetics of these metabolites. The re-usability of extraction columns was assessed in an attempt to reduce the cost of sample analysis. It was found that extraction columns could be used twice for the extraction of serum samples and up to four times for the extraction of urine samples. The difference between the re-usability of extraction columns for serum and urine samples was ascribed to various differences in the composition of the sample matrix. The stability of josamycin in stored serum and urine samples was also assessed.
- Full Text:
- Authors: Skinner, Michael Fredrick
- Date: 1992
- Subjects: Antibiotics -- Bioavailability , Antibiotics -- Pharmacokinetics
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3791 , http://hdl.handle.net/10962/d1003269
- Description: The investigations detailed herein have been conducted to address various aspects of the biopharmaceutics and pharmacokinetics of josamycin which to-date, have received little or no attention in the literature. Areas of investigation have included the selective determination of josamycin in serum and urine samples, the stability of josamycin in stored biological samples, intrinsic dissolution rates, solubility, acid and alkali stability and bioavailability and pharmacokinetics after dosing with a solution, powder and tablets. High performance liquid chromatography (HPLC) was used as the main analytical tool throughout these studies and proved to be highly versatile for the determination of josamycin in a number of different media. HPLC analysis afforded simple yet accurate determination of josamycin in samples from dissolution, solubility, tablet content and stability studies. Furthermore, the specificity afforded by HPLC was particularly useful for the separation of josamycin from degradation products formed in acid and alkali media. Since metabolites of josamycin are microbiologically active, microbiological assays do not determine the concentration solely of josamycin. An analytical method capable of the selective determination of josamycin in serum and urine samples is therefore required for the procurement of reliable bioavailability and pharmacokinetic data. HPLC affords this selectivity and a method for the selective determination of josamycin in serum and urine was successfully developed. The assay was simple yet precise, accurate and sensitive. Furthermore, it was well suited to the determination of josamycin in a large number of biological samples. Its success was largely due to the use of a solid phase extraction step using C₁₈ extraction columns, with a highly specific wash sequence followed by a phase separation step after elution from the extraction column. Chromatography was performed on a C₁₈ reversed-phase analytical column with UV detection of josamycin and internal standard at 231 nm and at 204 nm respectively using a programmable multi-wavelength detector. Only slight modification of the assay described should enable the selective determination of the metabolites of josamycin. This assay, therefore, lays the groundwork for future investigations into the pharmacokinetics of these metabolites. The re-usability of extraction columns was assessed in an attempt to reduce the cost of sample analysis. It was found that extraction columns could be used twice for the extraction of serum samples and up to four times for the extraction of urine samples. The difference between the re-usability of extraction columns for serum and urine samples was ascribed to various differences in the composition of the sample matrix. The stability of josamycin in stored serum and urine samples was also assessed.
- Full Text:
In vitro effects of three organic calcium channel blockers on the rat pineal gland
- Authors: Brown, Clint
- Date: 1992
- Subjects: Calcium -- Antagonists , Pineal gland -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3745 , http://hdl.handle.net/10962/d1003223 , Calcium -- Antagonists , Pineal gland -- Research
- Description: The calcium signal has emerged as an imponant component of intracellular regulation. Pineal function was thought to be slowed by the prominent calcification seen with increasing age, but recently it has been shown that calcium plays a crucial role in the adrenergic regulation of the gland. Beta-adrenoceptor stimulation increases melatonin (aMT) synthesis by increasing the activity of cyclic 3 '-5' adenosine mono phosphate (cAMP). Cyclic-AMP regulates the production of the pineal hormone, melatonin, from serotonin via the rate-limiting enzyme N-acetyltransferase (NAT). Increased intracellular cAMP is essential to the adrenergic induction of NAT. Noradrenaline(NA)also elevates pinealocyte cyclic guanosine monophosphate (cGMP). Adrenergic regulation of these cyclic nucleotides involves both α₁ - and β-adrenoceptors. Beta-adrenoceptor stimulation is an absolute requirement. Alphal-adrenoceptor activation, which is ineffective alone, serves to amplify the β-stimulated cAMP and cGMP responses via a positive effect on a Ca²⁺⁻/ phospholipiddependent protein kinase (Protein kinase-C) and a net influx of Ca²⁺ into the pinealocyte. Previous studies suggest the use of organic calcium channel blockers (CCBs) as probes of calcium-mediated processes. Applying this concept, the study set out to investigate the influence of a representative of each of the structurally diverse groups of calcium channel blockers viz. verapamil, diltiazem and nifedipine, and to examine their effect on β-adrenoceptor stimulation. It used the β-agonist isoprenaline (ISO) and the mixed [α₁/β]agonist noradrenaline (NA), for its combined [α₁/β]adrenoceptor stimulation, on agonist-induced increases in the production of radio-labelled aMT and N-acetylserotonin(aHT) -measured as the sum of N-acetylated product- from [¹⁴C] serotonin. This was done using organ cultures of rat pineal glands. It was speciously assumed that this drug paradigm would allow the determination of Ca²⁺ influx and/or the blocking thereof in the reported potentiation by using ISO as a non Ca²⁺ -entry stimulating agonist, compared with NA and its Ca²⁺ -entry stimulating properties. Surprisingly, all 3 CCB's potentiated the effect of NA. Only diltiazem was found not to potentiate the effect of ISO. In an attempt to uncover the reason for these results, the study moved toward a mechanistic approach,focusing in an antecedent manner on the various steps in the indole metabolic pathway to identify the point at which the change occurred, and hence possibly elucidate the mechanism responsible for the paradoxical increase. Experiments which assayed the levels of NAT, under the same drug conditions, showed the paradoxical increase to be already evident at this stage. Secondary experiments confirmed that NA stimulation of the pineal is dependent on Ca²⁺, both in organ culture and with NAT: the Ca²⁺ chelator EGTA abolished adrenergically-induced stimulation, while Ca²⁺ added after EGTA, restored the enzyme activity. The ionophore A23187 (which is able to transport Ca²⁺ directly into the pinealocyte via a mechanism which differs from the α₁ - mechanism) when used in conjunction with ISO or NA, was able to potentiate the responses of these two agonists relative to control values (agonist-alone), but by itself had no effect. With the enzyme NAT critically dependent upon cAMP for its induction, it was decided to determine the levels of cAMP and then those of its regulator, cAMP-phosphodiesterase (cAMP-PDE). This reasoning was prompted by reports of anti-calmodulin activity shown by the CCBs, in addition to their channel blocking effects. By binding to calmodulin (CaM), the CCBs are reportedly able to inhibit the CaM-dependent activation of cAMP-PDE. Following NA stimulation, verapamil caused a significant decrease in cAMP-PDE levels and an increase in cAMP. The other CCBs showed a similar trend. Glands stimulated with ISO in the presence of verapamil and nifedipine showed no significant differences in cAMP or cAMP-PDE levels. Diltiazem, however, was found to decrease the effect of ISO on cAMP while causing a concomitant increase in cAMP-PDE. This i) supported a possible hypothesis that the observed enhancement is a result of cAMP levels remaining elevated due to an inhibition of cAMP-PDE by the CCEs and ii) pointed to the possible presence of a CaM-sensitive PDE within the rat pineal gland. To test this hypothesis, two drugs which are more specific in their actions on CaM effects were chosen to see if the earlier results could be mimicked and thereby confirmed. Glands stimulated with NA in the presence of the specific CaM inhibitor R 24571 showed increased NAT activity and [¹⁴C]-aMT production. cAMP-PDE levels were clearly down, thus corroborating the possibility of cAMP-PDE inhibition. Glands incubated in the presence of M&B 22948, a CaM-sensitive PDE inhibitor, showed similar increases in NAT activity and [¹⁴C]-aMT. These findings therefore support the initial results and although indirect, confirm the hypothesis that the paradoxical increase following predominantly NA stimulation could be a result of cAMP levels remaining elevated, due to inhibition by the CCEs of the CaM-dependent activation of its regulator cAMP-PDE. In summary, data presented herein concur with proposals that: i) the CCEs are not specific enough to be used as tools to research Ca²⁺ -mediated events, as they appear to have sites of action other than the voltage operated channel (VOC); eg. binding to calmodulin, ii) there are functional differences between the CCEs as shown by diltiazem in this series of experiments, iii) there is a CaM-sensitive-PDE present in the pineal.
- Full Text:
- Authors: Brown, Clint
- Date: 1992
- Subjects: Calcium -- Antagonists , Pineal gland -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3745 , http://hdl.handle.net/10962/d1003223 , Calcium -- Antagonists , Pineal gland -- Research
- Description: The calcium signal has emerged as an imponant component of intracellular regulation. Pineal function was thought to be slowed by the prominent calcification seen with increasing age, but recently it has been shown that calcium plays a crucial role in the adrenergic regulation of the gland. Beta-adrenoceptor stimulation increases melatonin (aMT) synthesis by increasing the activity of cyclic 3 '-5' adenosine mono phosphate (cAMP). Cyclic-AMP regulates the production of the pineal hormone, melatonin, from serotonin via the rate-limiting enzyme N-acetyltransferase (NAT). Increased intracellular cAMP is essential to the adrenergic induction of NAT. Noradrenaline(NA)also elevates pinealocyte cyclic guanosine monophosphate (cGMP). Adrenergic regulation of these cyclic nucleotides involves both α₁ - and β-adrenoceptors. Beta-adrenoceptor stimulation is an absolute requirement. Alphal-adrenoceptor activation, which is ineffective alone, serves to amplify the β-stimulated cAMP and cGMP responses via a positive effect on a Ca²⁺⁻/ phospholipiddependent protein kinase (Protein kinase-C) and a net influx of Ca²⁺ into the pinealocyte. Previous studies suggest the use of organic calcium channel blockers (CCBs) as probes of calcium-mediated processes. Applying this concept, the study set out to investigate the influence of a representative of each of the structurally diverse groups of calcium channel blockers viz. verapamil, diltiazem and nifedipine, and to examine their effect on β-adrenoceptor stimulation. It used the β-agonist isoprenaline (ISO) and the mixed [α₁/β]agonist noradrenaline (NA), for its combined [α₁/β]adrenoceptor stimulation, on agonist-induced increases in the production of radio-labelled aMT and N-acetylserotonin(aHT) -measured as the sum of N-acetylated product- from [¹⁴C] serotonin. This was done using organ cultures of rat pineal glands. It was speciously assumed that this drug paradigm would allow the determination of Ca²⁺ influx and/or the blocking thereof in the reported potentiation by using ISO as a non Ca²⁺ -entry stimulating agonist, compared with NA and its Ca²⁺ -entry stimulating properties. Surprisingly, all 3 CCB's potentiated the effect of NA. Only diltiazem was found not to potentiate the effect of ISO. In an attempt to uncover the reason for these results, the study moved toward a mechanistic approach,focusing in an antecedent manner on the various steps in the indole metabolic pathway to identify the point at which the change occurred, and hence possibly elucidate the mechanism responsible for the paradoxical increase. Experiments which assayed the levels of NAT, under the same drug conditions, showed the paradoxical increase to be already evident at this stage. Secondary experiments confirmed that NA stimulation of the pineal is dependent on Ca²⁺, both in organ culture and with NAT: the Ca²⁺ chelator EGTA abolished adrenergically-induced stimulation, while Ca²⁺ added after EGTA, restored the enzyme activity. The ionophore A23187 (which is able to transport Ca²⁺ directly into the pinealocyte via a mechanism which differs from the α₁ - mechanism) when used in conjunction with ISO or NA, was able to potentiate the responses of these two agonists relative to control values (agonist-alone), but by itself had no effect. With the enzyme NAT critically dependent upon cAMP for its induction, it was decided to determine the levels of cAMP and then those of its regulator, cAMP-phosphodiesterase (cAMP-PDE). This reasoning was prompted by reports of anti-calmodulin activity shown by the CCBs, in addition to their channel blocking effects. By binding to calmodulin (CaM), the CCBs are reportedly able to inhibit the CaM-dependent activation of cAMP-PDE. Following NA stimulation, verapamil caused a significant decrease in cAMP-PDE levels and an increase in cAMP. The other CCBs showed a similar trend. Glands stimulated with ISO in the presence of verapamil and nifedipine showed no significant differences in cAMP or cAMP-PDE levels. Diltiazem, however, was found to decrease the effect of ISO on cAMP while causing a concomitant increase in cAMP-PDE. This i) supported a possible hypothesis that the observed enhancement is a result of cAMP levels remaining elevated due to an inhibition of cAMP-PDE by the CCEs and ii) pointed to the possible presence of a CaM-sensitive PDE within the rat pineal gland. To test this hypothesis, two drugs which are more specific in their actions on CaM effects were chosen to see if the earlier results could be mimicked and thereby confirmed. Glands stimulated with NA in the presence of the specific CaM inhibitor R 24571 showed increased NAT activity and [¹⁴C]-aMT production. cAMP-PDE levels were clearly down, thus corroborating the possibility of cAMP-PDE inhibition. Glands incubated in the presence of M&B 22948, a CaM-sensitive PDE inhibitor, showed similar increases in NAT activity and [¹⁴C]-aMT. These findings therefore support the initial results and although indirect, confirm the hypothesis that the paradoxical increase following predominantly NA stimulation could be a result of cAMP levels remaining elevated, due to inhibition by the CCEs of the CaM-dependent activation of its regulator cAMP-PDE. In summary, data presented herein concur with proposals that: i) the CCEs are not specific enough to be used as tools to research Ca²⁺ -mediated events, as they appear to have sites of action other than the voltage operated channel (VOC); eg. binding to calmodulin, ii) there are functional differences between the CCEs as shown by diltiazem in this series of experiments, iii) there is a CaM-sensitive-PDE present in the pineal.
- Full Text:
Neuropharmacological interactions in the rat pineal gland a study of antidepressant drugs
- Authors: Banoo, Shabir
- Date: 1992
- Subjects: Antidepressants -- Research , Pineal gland -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3744 , http://hdl.handle.net/10962/d1003222 , Antidepressants -- Research , Pineal gland -- Research
- Description: The rat pineal gland provides a convenient model for investigating nor adrenergic receptor neurotransmission and the effects of various drugs on these processes in health and disease. The effect of a variety of antidepressant drugs on rat pineal gland function following acute and chronic administration is described. Antidepressants from several different classes increase melatonin synthesis in rat pineal gland cultures when administered acutely. This effect appears to be mediated by noradrenaline acting on postsynaptic β-adrenoceptors. Activation of these receptors, in turn, activates the enzyme serotonin N-acetyltransferase via a cyclic adenosine monophosphate (cAMP) second messenger system. Serotonin N-acetyltransferase catalyses the rate-limiting conversion of serotonin to melatonin. Blockade of postsynaptic β-adrenoceptors prevents the antidepressant-induced increase in melatonin synthesis. The possibility that atypical antidepressants as well as those that selectively inhibit serotonin reuptake may increase melatonin synthesis via a β-adrenoceptor mechanism is discussed. In contrast, however, antidepressants from different classes have variable effects on rat pineal gland function when administered repeatedly. Chronic treatment with antidepressants that selectively inhibit noradrenaline reuptake appear to down-regulate the β-adrenoceptor system while, simultaneously, increasing melatonin output. Atypical antidepressants and those that selectively inhibit serotonin reuptake appear to be without these effects when administered repeatedly. The pineal gland of normal rats may therefore not represent a suitable model for evaluating the biochemical effects of chronic antidepressant treatment. In an attempt to investigatc pineal gland function in rats with "model depression" , antidepressants were administered to chronically reserpinized rats. Treatment with reserpine produced an increase in the density of pineal β-adrenoceptors. In addition, pineal cyclic AMP accumulation and N-acetyltransferase activity were increased in reserpinized rats following exogenous catecholamine stimulation. Reserpine, by depleting intraneuronal catecholamine stores, prevented the nocturnal induction of N-acetyltransferase activity and reduced the synthesis of melatonin in pineal gland cultures. A variety of antidepressants, irrespective of their acute pharmacological actions, reversed these effects when administered chronically to resepinized rats. Acute antidepressant administration was not associated with a reversal of the reserpine-induced effects. These findings provide additional evidence against the hypothesis that antidepressant drugs act by reducing noradrenergic neurotransmission and casts doubt on the importance of β-adrenoceptor down-regulation in the mechanism of antidepressant action. The possibility that the pineal gland of the reserpinized rat may represent an alternative model for evaluating antidepressant therapies is discussed.
- Full Text:
- Authors: Banoo, Shabir
- Date: 1992
- Subjects: Antidepressants -- Research , Pineal gland -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3744 , http://hdl.handle.net/10962/d1003222 , Antidepressants -- Research , Pineal gland -- Research
- Description: The rat pineal gland provides a convenient model for investigating nor adrenergic receptor neurotransmission and the effects of various drugs on these processes in health and disease. The effect of a variety of antidepressant drugs on rat pineal gland function following acute and chronic administration is described. Antidepressants from several different classes increase melatonin synthesis in rat pineal gland cultures when administered acutely. This effect appears to be mediated by noradrenaline acting on postsynaptic β-adrenoceptors. Activation of these receptors, in turn, activates the enzyme serotonin N-acetyltransferase via a cyclic adenosine monophosphate (cAMP) second messenger system. Serotonin N-acetyltransferase catalyses the rate-limiting conversion of serotonin to melatonin. Blockade of postsynaptic β-adrenoceptors prevents the antidepressant-induced increase in melatonin synthesis. The possibility that atypical antidepressants as well as those that selectively inhibit serotonin reuptake may increase melatonin synthesis via a β-adrenoceptor mechanism is discussed. In contrast, however, antidepressants from different classes have variable effects on rat pineal gland function when administered repeatedly. Chronic treatment with antidepressants that selectively inhibit noradrenaline reuptake appear to down-regulate the β-adrenoceptor system while, simultaneously, increasing melatonin output. Atypical antidepressants and those that selectively inhibit serotonin reuptake appear to be without these effects when administered repeatedly. The pineal gland of normal rats may therefore not represent a suitable model for evaluating the biochemical effects of chronic antidepressant treatment. In an attempt to investigatc pineal gland function in rats with "model depression" , antidepressants were administered to chronically reserpinized rats. Treatment with reserpine produced an increase in the density of pineal β-adrenoceptors. In addition, pineal cyclic AMP accumulation and N-acetyltransferase activity were increased in reserpinized rats following exogenous catecholamine stimulation. Reserpine, by depleting intraneuronal catecholamine stores, prevented the nocturnal induction of N-acetyltransferase activity and reduced the synthesis of melatonin in pineal gland cultures. A variety of antidepressants, irrespective of their acute pharmacological actions, reversed these effects when administered chronically to resepinized rats. Acute antidepressant administration was not associated with a reversal of the reserpine-induced effects. These findings provide additional evidence against the hypothesis that antidepressant drugs act by reducing noradrenergic neurotransmission and casts doubt on the importance of β-adrenoceptor down-regulation in the mechanism of antidepressant action. The possibility that the pineal gland of the reserpinized rat may represent an alternative model for evaluating antidepressant therapies is discussed.
- Full Text:
Structural analysis of some Escherichia coli capsular antigens
- Authors: Hackland, Peter Linton
- Date: 1992
- Subjects: Antigens , Enterobacteriaceae , Escherichia
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3758 , http://hdl.handle.net/10962/d1003236 , Antigens , Enterobacteriaceae , Escherichia
- Description: The work presented in this thesis forms part of a collaborative effort to determine the chemical structures of the surface antigens of bacteria which belong to the Enterobacteriaceae. These antigens are largely polysaccharides and occur as lipopolysaccharides and capsular polysaccharides which give rise to the somatic or 0 antigens and the capsular or K antigens, respectively. In recent years interest has mostly been focused on the extracellular polysaccharide antigens expressed by the genus Escherichia coli because of the effect they exert on normal immunological processes and their structural relatedness to the surface antigens of other more pathogenic bacteria. Therefore the molecular structures of the capsular polysaccharides (Kantigens)produced by E. coli 09:K35(AI04a) and 09:K38(A262a) have been determined by novel enzymic, chemical and spectroscopic procedures. These investigations show that the structures of these polysaccharides can be determined by a combination of chemical and spectroscopic procedures , or almost entirely by n.m.r. spectroscopy alone. The in vitro bacteriophage mediated depolymerisation of the native E. coli K35 polysaccharide demonstrates the value of this method for the isolation of oligosaccharides representing the repeating- unit and multiples thereof. Finally E. coli K37 and K38 capsular polysaccharides were used as model compounds for the evaluation of partial and selective reductive cleavage as methods of generating oligosaccharide for further structural analysis. The products of these reactions were analysed largely by a combination of mass spectrometric procedures.
- Full Text:
- Authors: Hackland, Peter Linton
- Date: 1992
- Subjects: Antigens , Enterobacteriaceae , Escherichia
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3758 , http://hdl.handle.net/10962/d1003236 , Antigens , Enterobacteriaceae , Escherichia
- Description: The work presented in this thesis forms part of a collaborative effort to determine the chemical structures of the surface antigens of bacteria which belong to the Enterobacteriaceae. These antigens are largely polysaccharides and occur as lipopolysaccharides and capsular polysaccharides which give rise to the somatic or 0 antigens and the capsular or K antigens, respectively. In recent years interest has mostly been focused on the extracellular polysaccharide antigens expressed by the genus Escherichia coli because of the effect they exert on normal immunological processes and their structural relatedness to the surface antigens of other more pathogenic bacteria. Therefore the molecular structures of the capsular polysaccharides (Kantigens)produced by E. coli 09:K35(AI04a) and 09:K38(A262a) have been determined by novel enzymic, chemical and spectroscopic procedures. These investigations show that the structures of these polysaccharides can be determined by a combination of chemical and spectroscopic procedures , or almost entirely by n.m.r. spectroscopy alone. The in vitro bacteriophage mediated depolymerisation of the native E. coli K35 polysaccharide demonstrates the value of this method for the isolation of oligosaccharides representing the repeating- unit and multiples thereof. Finally E. coli K37 and K38 capsular polysaccharides were used as model compounds for the evaluation of partial and selective reductive cleavage as methods of generating oligosaccharide for further structural analysis. The products of these reactions were analysed largely by a combination of mass spectrometric procedures.
- Full Text:
The evaluation of indomethacin and theophylline oral controlled/modified-release dosage forms in vitro-in vivo correlations
- Tandt, Ludo Alfons Germaan Luc
- Authors: Tandt, Ludo Alfons Germaan Luc
- Date: 1992
- Subjects: Theophylline , Indomethacin , Drugs -- Controlled release , Drugs -- Dosage forms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3794 , http://hdl.handle.net/10962/d1003272 , Theophylline , Indomethacin , Drugs -- Controlled release , Drugs -- Dosage forms
- Description: Over the past few decades many researchers have investigated the utility of in vitro - in vivo correlations for the assessment of dosage forms. These investigations are, however, dependent on reproducible dissolution data and well conducted biostudies in order to establish meaningful and robust correlations. Despite the fact that the establishment of such correlations is perhaps idealistic, considerable interest has still been shown in this area of research. Various Controlled/Modified Release Dosage Forms (CMRD's) of theophylline, a weakly basic drug, and indomethacin, a weakly acidic drug, were assessed in order to establish in vitro - in vivo correlations. Dissolution rate studies were carried out using either the USP basket or paddle apparatus. The dissolution rate studies were conducted in a range of dissolution media of varying pH. Bioavailability studies were conducted on the dosage forms used by the Biopharmaceutics Research Institute at Rhodes University. The results of these biostudies were kindly made available for use in this research project. Type A correlations were established using a mathematical simulation process whereby expected in vivo responses are simulated and compared to actual profiles obtained for the dosage forms. In order to perform the simulations the dissolution rate profiles were stripped and using linear regression and the methods of residuals the dissolution rate order and the relevant dissolution rates were obtained. The results of the s imulations indicated that the in vivo serum concentration-time curves could be accurately predicted for the theophylline dosage forms but to a lesser extent, for the indomethacin formulations. The dissolution rate studies indicated that the paddle method is a suitable method for dissolution rate studies of theophylline CMRD's, although it appeared that the optimum pH of the dissolution medium was formulation dependent. Dissolution rate studies conducted on indomethacin formulations indicated that the USP specified basket method for extended-release indomethacin formulations was not able to distinguish between two formulations which exhibited different in vivo profiles. The conversion to the paddle method was, however, able to highlight the differences between these formulations. The use of three dimensional topographs to depict dissolution rate profiles was demonstrated for formulations of both theophylline and indomethacin. The topographs enabled the successful differentiation between bioinequivalent formulations. The dissolution rate profiles were also fitted to the Wei bull equation and the parameters obtained from this were compared to the Weibull parameters obtained from the in vivo absorption plots obtained using the Wagner-Nelson method. The results indicated that the Weibull function was suitable to describe both the in vivo and in vitro data. The following recommendations for the preformulation dissolution studies of weakly acidic and weakly basic drugs are proposed. The dissolution rate studies of weakly acid drugs, such as indomethacin, should be carried out over a range of pH utilising the paddle apparatus. Three dimensional topographs based on the dissolution data should be constructed and used as a comparative tool for different formulations. Based on these comparisons the appropriate formulation can then be selected for a pilot scale in vivo bioavailability study. The dissolution rate studies of weakly basic drugs, such as theophylline, should be carried out over a range of pH utilising the paddle apparatus. The dissolution data should then be used to simulate the expected in vivo profile and on this basis the appropriate formulation selected for a pilot scale bioavailability study. The above approach to the preformulation studies of new CMRO's would allow for the more careful selection of new dosage forms and could thus eliminate costly and unnecessary bioavailability studies performed on inferior formulations.
- Full Text:
- Authors: Tandt, Ludo Alfons Germaan Luc
- Date: 1992
- Subjects: Theophylline , Indomethacin , Drugs -- Controlled release , Drugs -- Dosage forms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3794 , http://hdl.handle.net/10962/d1003272 , Theophylline , Indomethacin , Drugs -- Controlled release , Drugs -- Dosage forms
- Description: Over the past few decades many researchers have investigated the utility of in vitro - in vivo correlations for the assessment of dosage forms. These investigations are, however, dependent on reproducible dissolution data and well conducted biostudies in order to establish meaningful and robust correlations. Despite the fact that the establishment of such correlations is perhaps idealistic, considerable interest has still been shown in this area of research. Various Controlled/Modified Release Dosage Forms (CMRD's) of theophylline, a weakly basic drug, and indomethacin, a weakly acidic drug, were assessed in order to establish in vitro - in vivo correlations. Dissolution rate studies were carried out using either the USP basket or paddle apparatus. The dissolution rate studies were conducted in a range of dissolution media of varying pH. Bioavailability studies were conducted on the dosage forms used by the Biopharmaceutics Research Institute at Rhodes University. The results of these biostudies were kindly made available for use in this research project. Type A correlations were established using a mathematical simulation process whereby expected in vivo responses are simulated and compared to actual profiles obtained for the dosage forms. In order to perform the simulations the dissolution rate profiles were stripped and using linear regression and the methods of residuals the dissolution rate order and the relevant dissolution rates were obtained. The results of the s imulations indicated that the in vivo serum concentration-time curves could be accurately predicted for the theophylline dosage forms but to a lesser extent, for the indomethacin formulations. The dissolution rate studies indicated that the paddle method is a suitable method for dissolution rate studies of theophylline CMRD's, although it appeared that the optimum pH of the dissolution medium was formulation dependent. Dissolution rate studies conducted on indomethacin formulations indicated that the USP specified basket method for extended-release indomethacin formulations was not able to distinguish between two formulations which exhibited different in vivo profiles. The conversion to the paddle method was, however, able to highlight the differences between these formulations. The use of three dimensional topographs to depict dissolution rate profiles was demonstrated for formulations of both theophylline and indomethacin. The topographs enabled the successful differentiation between bioinequivalent formulations. The dissolution rate profiles were also fitted to the Wei bull equation and the parameters obtained from this were compared to the Weibull parameters obtained from the in vivo absorption plots obtained using the Wagner-Nelson method. The results indicated that the Weibull function was suitable to describe both the in vivo and in vitro data. The following recommendations for the preformulation dissolution studies of weakly acidic and weakly basic drugs are proposed. The dissolution rate studies of weakly acid drugs, such as indomethacin, should be carried out over a range of pH utilising the paddle apparatus. Three dimensional topographs based on the dissolution data should be constructed and used as a comparative tool for different formulations. Based on these comparisons the appropriate formulation can then be selected for a pilot scale in vivo bioavailability study. The dissolution rate studies of weakly basic drugs, such as theophylline, should be carried out over a range of pH utilising the paddle apparatus. The dissolution data should then be used to simulate the expected in vivo profile and on this basis the appropriate formulation selected for a pilot scale bioavailability study. The above approach to the preformulation studies of new CMRO's would allow for the more careful selection of new dosage forms and could thus eliminate costly and unnecessary bioavailability studies performed on inferior formulations.
- Full Text:
Design, development and evaluation of encapsulated oral controlled release theophylline mini-tablets
- Authors: Munday, Dale Leslie
- Date: 1991
- Subjects: Drugs -- Administration , Drugs -- Bioavailability , Drugs -- Controlled release , Drugs -- Dosage forms , Tablets (Medicine) , Biopharmaceutics , Drugs -- Testing
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3777 , http://hdl.handle.net/10962/d1003255 , Drugs -- Administration , Drugs -- Bioavailability , Drugs -- Controlled release , Drugs -- Dosage forms , Tablets (Medicine) , Biopharmaceutics , Drugs -- Testing
- Description: Conventional solid dosage forms often lead to fluctuations which exceed the maximum safe therapeutic level and/or decline below the minimum effective level. It is recognised that many drugs for chronic administration should be administered on a schedule that maintains plasma drug concentration within the therapeutic window. Research in controlled release dosage forms aims at designing a system with a zero-order input (eg, ideally to deliver 8.33% of the dose per hour over a 12 hour duration), producing steady state plasma drug levels. Oral dministration of drugs prepared as a controlled release formulation is extremely popular, and has attracted the attention of pharmaceutical scientists during the last decade. This has been due to the simultaneous convergence of various factors (eg, discovery of novel polymers and devices, better understanding of formulation and physiological constraints, expiration of existing patents, prohibitive cost of developing new drug entities), involved in the development of these delivery systems. Controlled release oral products can be formulated as single or multiple unit dosage forms and the relative merits of multiple unit forms with their own rate controlling systems are well established. This work describes the development of a relatively inexpensive multiple-unit capsule dosage form of theophylline containing coated mini-tablets for drug delivery throughout the gastrointestinal tract. Preformulation studies on theophylline anhydrous included solubility and dissolution rate determinations. Techniques including X-ray powder diffraction, differential scanning colorimetry and infrared spectroscopy provided no evidence of true polymorphism after recrystallisation from various solvents. However, scanning electron micrographs showed the effects of solvent polarity and cooling rate on the size and shape of recrystallised particles. Theophylline granules were manufactured by using various binders and were film coated by fluidised bed technology with various proportions of ethylcellulose, containing varying amounts of PEG 1540. In vitro release rates were dependent upon coating thickness and the proportion of PEG, which, being water soluble, created pores in the coating during dissolution studies as observed by a scanning electron microscope. However, substantial proportions of the drug remained unreleased from the granules. In order to overcome the problems of drug retention, plain granules were used and theophylline mini-tablets (3 mm diameter, weighing 15 - 20 mg) were manufactured and film coated with various Eudragits ® and other polymeric mixtures (soluble and insoluble). In vitro dissolution profiles from samples enclosed in hard gelatin capsules were determined using the USPXXI paddle apparatus in test media at pH 1.2 (HCI), pH 5.4 and 7.4 (phosphate buffers) at 37'C. Monitoring of in vitro theophylline release over 12 h, under identical hydrodynamic conditions, showed that the dissolution rate at pH 1.2 is substantially greater (95% of total drug content released in < 10 h) than that in phosphate buffers. The maximum release after 12 h was approximately 20 and 30% of total drug content of the tablet at pH 5.4 and 7.4, respectively. However, in vivo bioavailability after oral administration of tablets to rabbits corresponded to over 95% of total drug, compared with the same dose administered intravenously. The retarded drug release during in vitro dissolution in phosphate buffer was attributed to a possible interaction of phosphate ions with theophylline molecules at the tablet core-coat interface. These findings indicate that both rate and extent of theophylline release from the slow release coated mini-tablets are highly sensitive to phosphate buffers. The data also emphasise the usefulness of an animal model for assessment of in vivo drug release and subsequent absorption during the development of modified release dosage forms. Mini-tablets were subjected to isothermal and cyclic stresses to reach conditions for up to 6 months at different temperatures and relative humidity. The film integrity was maintained but ageing of the coating occurred which impeded dissolution. Reduced drug release was temperature related while the effect of relative humidi% was insignific~t. Encapsulated mini-tablets (uncoated and coated with Eudragit RL and RS 2% w/w) equivalent to a 300 mg dose, were evaluated both in vitro and in vivo using beagle dogs. The pharmacokinetic parameters from single and multiple dose studies showed several advantages over Theo-Dur® 300 mg tablets. Precise dosage titration is possible by careful adjustment of the number of encapsulated mini-tablets. This multiple unit mini-tablet delivery system will allow for greater flexibility in dosage adjustment compared to the currently available preparations, allowing individualised fine dose titration in those patients requiring therapeutic drug monitoring. The developmentof the multiple unit mini-tablet formulation appears to provide an optimal dosage form with maximum flexibility in respect of dose, duration range and ease of production.
- Full Text:
Design, development and evaluation of encapsulated oral controlled release theophylline mini-tablets
- Authors: Munday, Dale Leslie
- Date: 1991
- Subjects: Drugs -- Administration , Drugs -- Bioavailability , Drugs -- Controlled release , Drugs -- Dosage forms , Tablets (Medicine) , Biopharmaceutics , Drugs -- Testing
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3777 , http://hdl.handle.net/10962/d1003255 , Drugs -- Administration , Drugs -- Bioavailability , Drugs -- Controlled release , Drugs -- Dosage forms , Tablets (Medicine) , Biopharmaceutics , Drugs -- Testing
- Description: Conventional solid dosage forms often lead to fluctuations which exceed the maximum safe therapeutic level and/or decline below the minimum effective level. It is recognised that many drugs for chronic administration should be administered on a schedule that maintains plasma drug concentration within the therapeutic window. Research in controlled release dosage forms aims at designing a system with a zero-order input (eg, ideally to deliver 8.33% of the dose per hour over a 12 hour duration), producing steady state plasma drug levels. Oral dministration of drugs prepared as a controlled release formulation is extremely popular, and has attracted the attention of pharmaceutical scientists during the last decade. This has been due to the simultaneous convergence of various factors (eg, discovery of novel polymers and devices, better understanding of formulation and physiological constraints, expiration of existing patents, prohibitive cost of developing new drug entities), involved in the development of these delivery systems. Controlled release oral products can be formulated as single or multiple unit dosage forms and the relative merits of multiple unit forms with their own rate controlling systems are well established. This work describes the development of a relatively inexpensive multiple-unit capsule dosage form of theophylline containing coated mini-tablets for drug delivery throughout the gastrointestinal tract. Preformulation studies on theophylline anhydrous included solubility and dissolution rate determinations. Techniques including X-ray powder diffraction, differential scanning colorimetry and infrared spectroscopy provided no evidence of true polymorphism after recrystallisation from various solvents. However, scanning electron micrographs showed the effects of solvent polarity and cooling rate on the size and shape of recrystallised particles. Theophylline granules were manufactured by using various binders and were film coated by fluidised bed technology with various proportions of ethylcellulose, containing varying amounts of PEG 1540. In vitro release rates were dependent upon coating thickness and the proportion of PEG, which, being water soluble, created pores in the coating during dissolution studies as observed by a scanning electron microscope. However, substantial proportions of the drug remained unreleased from the granules. In order to overcome the problems of drug retention, plain granules were used and theophylline mini-tablets (3 mm diameter, weighing 15 - 20 mg) were manufactured and film coated with various Eudragits ® and other polymeric mixtures (soluble and insoluble). In vitro dissolution profiles from samples enclosed in hard gelatin capsules were determined using the USPXXI paddle apparatus in test media at pH 1.2 (HCI), pH 5.4 and 7.4 (phosphate buffers) at 37'C. Monitoring of in vitro theophylline release over 12 h, under identical hydrodynamic conditions, showed that the dissolution rate at pH 1.2 is substantially greater (95% of total drug content released in < 10 h) than that in phosphate buffers. The maximum release after 12 h was approximately 20 and 30% of total drug content of the tablet at pH 5.4 and 7.4, respectively. However, in vivo bioavailability after oral administration of tablets to rabbits corresponded to over 95% of total drug, compared with the same dose administered intravenously. The retarded drug release during in vitro dissolution in phosphate buffer was attributed to a possible interaction of phosphate ions with theophylline molecules at the tablet core-coat interface. These findings indicate that both rate and extent of theophylline release from the slow release coated mini-tablets are highly sensitive to phosphate buffers. The data also emphasise the usefulness of an animal model for assessment of in vivo drug release and subsequent absorption during the development of modified release dosage forms. Mini-tablets were subjected to isothermal and cyclic stresses to reach conditions for up to 6 months at different temperatures and relative humidity. The film integrity was maintained but ageing of the coating occurred which impeded dissolution. Reduced drug release was temperature related while the effect of relative humidi% was insignific~t. Encapsulated mini-tablets (uncoated and coated with Eudragit RL and RS 2% w/w) equivalent to a 300 mg dose, were evaluated both in vitro and in vivo using beagle dogs. The pharmacokinetic parameters from single and multiple dose studies showed several advantages over Theo-Dur® 300 mg tablets. Precise dosage titration is possible by careful adjustment of the number of encapsulated mini-tablets. This multiple unit mini-tablet delivery system will allow for greater flexibility in dosage adjustment compared to the currently available preparations, allowing individualised fine dose titration in those patients requiring therapeutic drug monitoring. The developmentof the multiple unit mini-tablet formulation appears to provide an optimal dosage form with maximum flexibility in respect of dose, duration range and ease of production.
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Structural studies on the capsular antigens of Escherichia coli serotypes K102 and K47
- Authors: De Bruin, Aletta Hester
- Date: 1991
- Subjects: Escherichia -- Research , Klebsiella -- Research , Enterobacteriaceae -- Research , Antigens -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3754 , http://hdl.handle.net/10962/d1003232 , Escherichia -- Research , Klebsiella -- Research , Enterobacteriaceae -- Research , Antigens -- Research
- Description: The work presented in this thesis forms part of a continuing programme concerned with the structural determination of capsular polysaccharides of some Enterobacteriaceae. Since bacteria of this family are pathogenic to Man, work in this laboratory has focused on the structural elucidation of Klebsiella and, more recently, Escherichia coli ( E. coli) capsular polysaccharides ( K-antigens ). To date, some 74 K-antigens have been distinguished serologically within the genus E. coli and the structures of approximately 70% are known. In general, the K-antigens of the E. coli are characterized by a wide variety of constituent monosaccharides arranged in repeating units. In this thesis the structural elucidation of the capsular polysaccharides of E. coli serotypes 08: KI02: H- and 08: K47: H2 is presented. A variety of chemical techniques has been employed in the structural analysis, and are discussed. The thesis also includes extensive two-dimensional n.m.r. studies on the E. coli KI02 and K47 polysaccharides, as well as on a modified KI02 polymer produced after a lithium-ethylenediamine degradation of the native polysaccharide.
- Full Text:
- Authors: De Bruin, Aletta Hester
- Date: 1991
- Subjects: Escherichia -- Research , Klebsiella -- Research , Enterobacteriaceae -- Research , Antigens -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3754 , http://hdl.handle.net/10962/d1003232 , Escherichia -- Research , Klebsiella -- Research , Enterobacteriaceae -- Research , Antigens -- Research
- Description: The work presented in this thesis forms part of a continuing programme concerned with the structural determination of capsular polysaccharides of some Enterobacteriaceae. Since bacteria of this family are pathogenic to Man, work in this laboratory has focused on the structural elucidation of Klebsiella and, more recently, Escherichia coli ( E. coli) capsular polysaccharides ( K-antigens ). To date, some 74 K-antigens have been distinguished serologically within the genus E. coli and the structures of approximately 70% are known. In general, the K-antigens of the E. coli are characterized by a wide variety of constituent monosaccharides arranged in repeating units. In this thesis the structural elucidation of the capsular polysaccharides of E. coli serotypes 08: KI02: H- and 08: K47: H2 is presented. A variety of chemical techniques has been employed in the structural analysis, and are discussed. The thesis also includes extensive two-dimensional n.m.r. studies on the E. coli KI02 and K47 polysaccharides, as well as on a modified KI02 polymer produced after a lithium-ethylenediamine degradation of the native polysaccharide.
- Full Text:
A study of possible interactions between the pineal gland and the opioidergic system
- Authors: Khan, Razeeya B
- Date: 1990
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3729 , http://hdl.handle.net/10962/d1001468
- Description: Recent observations suggest a link between the pineal gland and the opioid system. Possible areas of interaction between the pineal gland and the opioidergic system in Wistar rats were investigated. The effect of opioids on the pineal gland in organ culture was monitored. Neither morphine, methadone nor the opioid antagonist naloxone was found to affect [¹⁴C]-serotonin metabolism by the pineal gland in vitro. Both the pineal gland and the opioid system are influenced by exposure to stressful stimuli. Morphine and melatonin had protective effects on stress-induced gastric lesions. The ability of melatonin to inhibit lesion formation was found not to be exerted via an opioidergic mechanism. Evidence has been obtained for a possible modulation of the stress response by the pineal gland . The opioid drugs are the most potent analgesic agents available. A possible interaction between the opioid system and the pineal gland in the modulation of the response to noxious stimuli was investigated. An intact pineal gland was found to be necessary for the manifestation of the nocturnally increased response of rats to noxious stimuli
- Full Text:
- Authors: Khan, Razeeya B
- Date: 1990
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3729 , http://hdl.handle.net/10962/d1001468
- Description: Recent observations suggest a link between the pineal gland and the opioid system. Possible areas of interaction between the pineal gland and the opioidergic system in Wistar rats were investigated. The effect of opioids on the pineal gland in organ culture was monitored. Neither morphine, methadone nor the opioid antagonist naloxone was found to affect [¹⁴C]-serotonin metabolism by the pineal gland in vitro. Both the pineal gland and the opioid system are influenced by exposure to stressful stimuli. Morphine and melatonin had protective effects on stress-induced gastric lesions. The ability of melatonin to inhibit lesion formation was found not to be exerted via an opioidergic mechanism. Evidence has been obtained for a possible modulation of the stress response by the pineal gland . The opioid drugs are the most potent analgesic agents available. A possible interaction between the opioid system and the pineal gland in the modulation of the response to noxious stimuli was investigated. An intact pineal gland was found to be necessary for the manifestation of the nocturnally increased response of rats to noxious stimuli
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A study of the effects of the pineal hormone, melatonin, on dopaminergic transmission in the central nervous system of rats
- Authors: Burton, Susan Frances
- Date: 1990
- Subjects: Dopaminergic mechanisms Melatonin Pineal gland -- Secretions Neural transmission Pineal gland Nervous system
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3726 , http://hdl.handle.net/10962/d1001463
- Description: Dopamine mechanisms in the central nervous system are important in the control of both normal and abnormal motor function. The recent observations in both animal and human studies, that melatonin, the principal hormone of the pineal gland, may have a role in the control of movement and the pathophysiology of movement disorders, have given rise to the concept that melatonin may have a modulatory influence on central dopaminergic neurotransmission. This study makes use of three animal behavioural models as well as a biochemical model of central dopaminergic function to further investigate the concept. Results from studies using the biochemical model, which investigated the effect of melatonin on dopamine and apomorphine stimulation of dopamine-sensitive adenylate cylase, suggest that melatonin is neither a competitive antagonist nor agonist at the D₁ receptor level, although the possibility of physiological stimulation or antagonism is not excluded. In behavioural studies, prior melatonin mg/kg administration (1 and 10 (8M) ip) inhibited apomorphine induced stereotypy and locomotor activity in normal rats, and apomorphine-induced rotational behaviour in 6-hydroxydopamine and quinolinic acid lesioned rats. The possibility that these results may have physiological significance is borne out by the observation that, under enviromental lighting conditions that are associated with raised endogeous melatonin levels, apomorphine- induced stereotypy and locomotor activity is attenuated. The general conclusion is that melatonin has an inhibitory influence on central nervous system dopaminergic function, suggesting therefore, that the pineal gland and melatonin may have a role in the pathophysiology and treatment of movement and behavioural disorders associated with dopaminergic dysfunction
- Full Text:
- Authors: Burton, Susan Frances
- Date: 1990
- Subjects: Dopaminergic mechanisms Melatonin Pineal gland -- Secretions Neural transmission Pineal gland Nervous system
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3726 , http://hdl.handle.net/10962/d1001463
- Description: Dopamine mechanisms in the central nervous system are important in the control of both normal and abnormal motor function. The recent observations in both animal and human studies, that melatonin, the principal hormone of the pineal gland, may have a role in the control of movement and the pathophysiology of movement disorders, have given rise to the concept that melatonin may have a modulatory influence on central dopaminergic neurotransmission. This study makes use of three animal behavioural models as well as a biochemical model of central dopaminergic function to further investigate the concept. Results from studies using the biochemical model, which investigated the effect of melatonin on dopamine and apomorphine stimulation of dopamine-sensitive adenylate cylase, suggest that melatonin is neither a competitive antagonist nor agonist at the D₁ receptor level, although the possibility of physiological stimulation or antagonism is not excluded. In behavioural studies, prior melatonin mg/kg administration (1 and 10 (8M) ip) inhibited apomorphine induced stereotypy and locomotor activity in normal rats, and apomorphine-induced rotational behaviour in 6-hydroxydopamine and quinolinic acid lesioned rats. The possibility that these results may have physiological significance is borne out by the observation that, under enviromental lighting conditions that are associated with raised endogeous melatonin levels, apomorphine- induced stereotypy and locomotor activity is attenuated. The general conclusion is that melatonin has an inhibitory influence on central nervous system dopaminergic function, suggesting therefore, that the pineal gland and melatonin may have a role in the pathophysiology and treatment of movement and behavioural disorders associated with dopaminergic dysfunction
- Full Text:
Chemical and spectroscopic studies of the capsular polysaccharides of some klebsiella and escherichia coli serotypes
- Authors: Stanley, Shawn Mark Ross
- Date: 1990
- Subjects: Polysaccharides , Klebsiella , Escherichia
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3736 , http://hdl.handle.net/10962/d1001525
- Description: The work described in this thesis forms part of an international programme concerned with the structure elucidation of the capsular antigens of some Enterobacteriaceae. Many of the Klebsiella and some of the Escherichia coli are pathogenic to man and, hence, they are of interest. The virulence of bacteria is a multifactorial phenomenon, in which characteristic traits of bacteria and their hosts play comparable and complementary roles. It is accepted that pathogens are more virulent when encapsulated, because, nearly all disease causing bacteria have a capsule when freshly isolated from the host. This increase in pathogenicity is related, in part, to the capsular polysaccharides' ability to avoid or attenuate the host defence mechanisms. In the majority of cases the protective aspects of the capsule are overcome in the latter stages of infection when the formation of specific antibodies by the host has occurred. However there are situations in which an immune state of the infected host is virtually never reached, and susceptiblity to the infecting bacteria is maintained even in the advanced stage of an infection. Explanation of this phenomenon becomes possible by analysing the structure of the polysaccharides. The inability of the host to raise an immune response to the capsule may be because the structure of the polysaccharide is similar or identical to the host's carbohydrates. The serological and pathogenic relatedness of encapsulated E. coli and Klebsiella, to the encapsulated strains of other genera, is based on structural identity or similarity of the respective capsules. Capsular polysaccharides are analysed by both chemical and instrumental methods, and, at present, nuclear magnetic resonance spectroscopy is the most important analytical technique
- Full Text:
- Authors: Stanley, Shawn Mark Ross
- Date: 1990
- Subjects: Polysaccharides , Klebsiella , Escherichia
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3736 , http://hdl.handle.net/10962/d1001525
- Description: The work described in this thesis forms part of an international programme concerned with the structure elucidation of the capsular antigens of some Enterobacteriaceae. Many of the Klebsiella and some of the Escherichia coli are pathogenic to man and, hence, they are of interest. The virulence of bacteria is a multifactorial phenomenon, in which characteristic traits of bacteria and their hosts play comparable and complementary roles. It is accepted that pathogens are more virulent when encapsulated, because, nearly all disease causing bacteria have a capsule when freshly isolated from the host. This increase in pathogenicity is related, in part, to the capsular polysaccharides' ability to avoid or attenuate the host defence mechanisms. In the majority of cases the protective aspects of the capsule are overcome in the latter stages of infection when the formation of specific antibodies by the host has occurred. However there are situations in which an immune state of the infected host is virtually never reached, and susceptiblity to the infecting bacteria is maintained even in the advanced stage of an infection. Explanation of this phenomenon becomes possible by analysing the structure of the polysaccharides. The inability of the host to raise an immune response to the capsule may be because the structure of the polysaccharide is similar or identical to the host's carbohydrates. The serological and pathogenic relatedness of encapsulated E. coli and Klebsiella, to the encapsulated strains of other genera, is based on structural identity or similarity of the respective capsules. Capsular polysaccharides are analysed by both chemical and instrumental methods, and, at present, nuclear magnetic resonance spectroscopy is the most important analytical technique
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A critical evaluation of the human skin blanching assay and comparative bioavailability studies on topical corticosteroid preparations
- Authors: Meyer, Eric
- Date: 1989
- Subjects: Dermatopharmacology Skin, Effect of drugs on Adrenocortical hormones
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3727 , http://hdl.handle.net/10962/d1001464
- Description: Several aspects of the human skin blanching assay were evaluated in an attempt to suggest improvements in the methodology of this assay. Three trials were performed in the unoccluded application mode, using two proprietary creams containing 0,1% betamethasone (as the 17-valerate). Preliminary observations of the influence of ambient temperature and relative humidity on the blanching response did not allow definite conclusions to be drawn. Studies on the number of observers required for reliable results of comparative blanching indicated that at least two trained observers should be employed. Analyses of the results of individual volunteers demonstrated the expected biological variability, and suggest that subjects selected for trials should represent a range of blanching responses. No sex-related differences in blanching responses were found, and both arms exhibited similar sensitivity to corticosteroids. Retrospective analysis of 95 040 observations of blanching responses showed that in the unoccluded application mode blanching is lowest close to the wrist, and in the occluded mode blanching is lowest close to the elbow. Studies on the method of transportation of Betnovate preparations suggest that topical formulations should not be exposed to temperature extremes during transportation. It is proposed that patients should not transport topical formulations in the holds of ships or aircraft, and that exporters and manufacturers should make use of special transportation and storage conditions. In a study of ten topical formulations from three countries it was found that there was no trend of products from one country consistently exhibiting superior blanching to products from the other two countries, or products from one country consistently exhibiting the lowest degree of blanching, although considerable differences in blanching responses were found in some cases. Interpretation of the results of these studies demonstrated the importance of employing a combination of statistical analyses, blanching profiles and AUC values when drawing conclusions regarding comparative bioavailability. A study of the blanching profiles of Betnovate cream included in all 16 trials performed during this work indicated that this preparation behaved in a similar fashion during all trials, thereby giving credence to the results of the trials
- Full Text:
- Authors: Meyer, Eric
- Date: 1989
- Subjects: Dermatopharmacology Skin, Effect of drugs on Adrenocortical hormones
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3727 , http://hdl.handle.net/10962/d1001464
- Description: Several aspects of the human skin blanching assay were evaluated in an attempt to suggest improvements in the methodology of this assay. Three trials were performed in the unoccluded application mode, using two proprietary creams containing 0,1% betamethasone (as the 17-valerate). Preliminary observations of the influence of ambient temperature and relative humidity on the blanching response did not allow definite conclusions to be drawn. Studies on the number of observers required for reliable results of comparative blanching indicated that at least two trained observers should be employed. Analyses of the results of individual volunteers demonstrated the expected biological variability, and suggest that subjects selected for trials should represent a range of blanching responses. No sex-related differences in blanching responses were found, and both arms exhibited similar sensitivity to corticosteroids. Retrospective analysis of 95 040 observations of blanching responses showed that in the unoccluded application mode blanching is lowest close to the wrist, and in the occluded mode blanching is lowest close to the elbow. Studies on the method of transportation of Betnovate preparations suggest that topical formulations should not be exposed to temperature extremes during transportation. It is proposed that patients should not transport topical formulations in the holds of ships or aircraft, and that exporters and manufacturers should make use of special transportation and storage conditions. In a study of ten topical formulations from three countries it was found that there was no trend of products from one country consistently exhibiting superior blanching to products from the other two countries, or products from one country consistently exhibiting the lowest degree of blanching, although considerable differences in blanching responses were found in some cases. Interpretation of the results of these studies demonstrated the importance of employing a combination of statistical analyses, blanching profiles and AUC values when drawing conclusions regarding comparative bioavailability. A study of the blanching profiles of Betnovate cream included in all 16 trials performed during this work indicated that this preparation behaved in a similar fashion during all trials, thereby giving credence to the results of the trials
- Full Text:
Application of high-performance liquid chromatography for the analysis and pharmocokinetics of mephenoxalone
- Authors: Van der Westhuizen, Fiona
- Date: 1988 , 2013-03-06
- Subjects: High performance liquid chromatography , Central nervous system depressants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3810 , http://hdl.handle.net/10962/d1004385 , High performance liquid chromatography , Central nervous system depressants
- Description: Mephenoxalone is a mild central nervous system depressant with activity resembling that of meprobamate. Since its introduction in 1961 mephenoxalone has been used as an anxiolytic and as a muscle relaxant, although the latter effect is weak. Preliminary studies on the absorption and disposition of mephenoxalone have been conducted in beagle dogs but no pharmacokinetic data from human studies have been reported, except for a single study in which the biotransformation products present in human urine were identified. Methods presently available for the determination of mephenoxalone in biological fluids lack the sensitivity, specificity and precision required for detailed pharmacokinetic studies. In this study, a rapid, sensitive, precise reverse-phase high-performance liquid chromatographic method with ultraviolet detection at 200nm was employed for the determination of mephenoxalone in biological fluids. Serum and urine samples were prepared for chromatographic analysis using simple liquid-liquid extraction techniques. The application of the assay to pharmacokinetic studies in humans is presented. After administration of a single oral dose of 400mg mephenoxalone dispersed in 150ml water to six young, healthy volunteers, the compound was rapidly absorbed with the peak concentration of 8μg/ml occurring after about 1 hour. The elimination half-life was approximately 3 hours. The drug was extensively metabolized with only about 1 percent of the administered dose being excreted unchanged in the urine after 24 hours. The bioavailability of a newly developed mephenoxalone-containing tablet was also investigated. The drug was absorbed more rapidly from the tablet than from the dispersed dose. This was attributed to a shorter in vivo dissolution time on the basis of in vitro tests, but this effect is not expected to be clinically significant. In addition, two human urinary metabolites of mephenoxalone were identified as unconjugated hydroxylated derivatives using thermospray HPLC-mass spectrometry. The plasma protein-binding properties of mephenoxalone were also investigated.
- Full Text:
- Authors: Van der Westhuizen, Fiona
- Date: 1988 , 2013-03-06
- Subjects: High performance liquid chromatography , Central nervous system depressants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3810 , http://hdl.handle.net/10962/d1004385 , High performance liquid chromatography , Central nervous system depressants
- Description: Mephenoxalone is a mild central nervous system depressant with activity resembling that of meprobamate. Since its introduction in 1961 mephenoxalone has been used as an anxiolytic and as a muscle relaxant, although the latter effect is weak. Preliminary studies on the absorption and disposition of mephenoxalone have been conducted in beagle dogs but no pharmacokinetic data from human studies have been reported, except for a single study in which the biotransformation products present in human urine were identified. Methods presently available for the determination of mephenoxalone in biological fluids lack the sensitivity, specificity and precision required for detailed pharmacokinetic studies. In this study, a rapid, sensitive, precise reverse-phase high-performance liquid chromatographic method with ultraviolet detection at 200nm was employed for the determination of mephenoxalone in biological fluids. Serum and urine samples were prepared for chromatographic analysis using simple liquid-liquid extraction techniques. The application of the assay to pharmacokinetic studies in humans is presented. After administration of a single oral dose of 400mg mephenoxalone dispersed in 150ml water to six young, healthy volunteers, the compound was rapidly absorbed with the peak concentration of 8μg/ml occurring after about 1 hour. The elimination half-life was approximately 3 hours. The drug was extensively metabolized with only about 1 percent of the administered dose being excreted unchanged in the urine after 24 hours. The bioavailability of a newly developed mephenoxalone-containing tablet was also investigated. The drug was absorbed more rapidly from the tablet than from the dispersed dose. This was attributed to a shorter in vivo dissolution time on the basis of in vitro tests, but this effect is not expected to be clinically significant. In addition, two human urinary metabolites of mephenoxalone were identified as unconjugated hydroxylated derivatives using thermospray HPLC-mass spectrometry. The plasma protein-binding properties of mephenoxalone were also investigated.
- Full Text:
Application of high-performance liquid chromatography to the analysis, stability and pharmacokinetics of erythromycin
- Authors: Stubbs, Christopher
- Date: 1988
- Subjects: Erythromycin High performance liquid chromatography
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3809 , http://hdl.handle.net/10962/d1004372
- Description: Erythromycin is a macrolide antibiotic used mainly in the treatment of infections caused by gram-positive organisms. Erythromycin base is rap idly degraded in acidic media necessitating the use of structurally modified erythromycin derivatives or acid resistant dosage forms in order to decrease gastric inactivation of the drug. The majority of pharmacokinetic studies to-date have utilized relatively non-specific microbiological assay procedures which are unable to differentiate between concentrations of active erythromycin base and the inactive pro-drug derivatives. A high-performance liquid chromatographic (HPLC) technique is described for the simultaneous determination of erythromycin base and propionate (inactive pro-drug form) in human serum and urine following the oral administration of erythromycin estolate, an acid stable derivative of erythromycin. The method involves a solid-phase extraction step prior to chromatography on a C18 reversed-phase column with coulometric electrochemical detection. Sample handling and storage techniques are presented which minimize hydrolysis of the inactive ester moiety between sample collection and analysis, thereby more accurately reflecting the in vivo situation than in previously published studies. Results from single dose pharmacokinetic studies indicate that only 10-15% of the total erythromycin concentration in vivo is present as the active base component following oral administration of erythromycin estolate. This percentage increases to approximately 25% during multiple dose administration. Novel urinary excretion data are presented which reveal that approximately 40% and 55% of the total erythromycin excreted in urine is excreted as erythromycin base following single and multiple dosages respectively. Computer fitting of mean serum concentration-time data revealed that an open one compartment model with linear first order absorption and elimination best described the absorption and disposition of erythromycin, although poor computer fits for individual data sets were observed. Some evidence of non-linear elimination is presented utilizing both compartmental and non-compartmental pharmacokinetic techniques. Large intra-and inter-personal variability in erythromycin absorption and disposition was experienced which was evaluated in five subjects who each received one 500 mg erythromycin estolate tablet from the same batch, on three separate occasions. In addition. an HPLC method is described for the analysis of "total erythromycin" concentrations following erythromycin estolate administration which involves hydrolysis of the ester component prior to chromatography. as well as an HPLC method utilizing amperometric electrochemical detection capable of monitoring the stability of erythromycin base in stored biological fluids. These methods were uti I ized in various stability studies involving erythromycin base and propionate as well as for the analysis of erythromycin estolate dosage forms.
- Full Text:
- Authors: Stubbs, Christopher
- Date: 1988
- Subjects: Erythromycin High performance liquid chromatography
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3809 , http://hdl.handle.net/10962/d1004372
- Description: Erythromycin is a macrolide antibiotic used mainly in the treatment of infections caused by gram-positive organisms. Erythromycin base is rap idly degraded in acidic media necessitating the use of structurally modified erythromycin derivatives or acid resistant dosage forms in order to decrease gastric inactivation of the drug. The majority of pharmacokinetic studies to-date have utilized relatively non-specific microbiological assay procedures which are unable to differentiate between concentrations of active erythromycin base and the inactive pro-drug derivatives. A high-performance liquid chromatographic (HPLC) technique is described for the simultaneous determination of erythromycin base and propionate (inactive pro-drug form) in human serum and urine following the oral administration of erythromycin estolate, an acid stable derivative of erythromycin. The method involves a solid-phase extraction step prior to chromatography on a C18 reversed-phase column with coulometric electrochemical detection. Sample handling and storage techniques are presented which minimize hydrolysis of the inactive ester moiety between sample collection and analysis, thereby more accurately reflecting the in vivo situation than in previously published studies. Results from single dose pharmacokinetic studies indicate that only 10-15% of the total erythromycin concentration in vivo is present as the active base component following oral administration of erythromycin estolate. This percentage increases to approximately 25% during multiple dose administration. Novel urinary excretion data are presented which reveal that approximately 40% and 55% of the total erythromycin excreted in urine is excreted as erythromycin base following single and multiple dosages respectively. Computer fitting of mean serum concentration-time data revealed that an open one compartment model with linear first order absorption and elimination best described the absorption and disposition of erythromycin, although poor computer fits for individual data sets were observed. Some evidence of non-linear elimination is presented utilizing both compartmental and non-compartmental pharmacokinetic techniques. Large intra-and inter-personal variability in erythromycin absorption and disposition was experienced which was evaluated in five subjects who each received one 500 mg erythromycin estolate tablet from the same batch, on three separate occasions. In addition. an HPLC method is described for the analysis of "total erythromycin" concentrations following erythromycin estolate administration which involves hydrolysis of the ester component prior to chromatography. as well as an HPLC method utilizing amperometric electrochemical detection capable of monitoring the stability of erythromycin base in stored biological fluids. These methods were uti I ized in various stability studies involving erythromycin base and propionate as well as for the analysis of erythromycin estolate dosage forms.
- Full Text:
Aspects of the transdermal permeation and analysis of betamethasone 17-valerate
- Authors: Smith, Eric W
- Date: 1988
- Subjects: Transdermal medication Skin absorption Dermatologic agents
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3815 , http://hdl.handle.net/10962/d1004741
- Description: The current world-wide interest in transdermal drug delivery makes the prospect of valid in vitro diffusion cell methodology highly attractive. A new laboratory diffusion cell has been designed and constructed based on theoretical principles and practical permeation reports surveyed in recent literature, and has been applied to the monitoring of betamethasone 17-valerate permeation. The cell performance has been validated with respect to hydrodynamic mixing efficiency and temperature of the receptor phase. The steady-state permeation of this corticosteroid has been monitored through various synthetic and animal membranes in order to select the most appropriate media for in vitro study. The permeation of betamethasone 17-valerate has been monitored from various types of commercial and extemporaneously prepared semisolid topical formulation (cream, lotion, ointment and scalp application), through silicone membrane, human and weanling pig stratum corneum, and full thickness hairless mouse skin, and these in vitro results have been compared to data from in vivo blanching assays, using the same formulations, in an attempt to correlate the findings. This experimental methodology has necessitated the development of ancillary analytical techniques. A column-switching high-performance liquid chromatographic method has been developed for the rapid on-line clean-up and analysis of betamethasone 17-valerate contained in the various topical formulations, which minimizes sample handling and extraction procedures. The method has been modified for the analysis of this corticosteroid in the isopropyl myristate receptor phase used in the in vitro permeation experiments, and scintillation counting of tritium-labelled water has been used to verify the integrity of the animal membranes. The comparison of in vitro permeation and in vivo blanching results indicate good correlation of the data in certain instances. The closest correlations have been observed when the human stratum corneum has been used in vitro and these results are compared to data from the occluded mode of the blanching assay. The results of the porcine and murine media have also correlated with the human in vivo data, whereas the silicone membrane appears applicable only in certain in vitro experiments. The results indicate that valid, comparative percutaneous absorption data may be obtained in vitro by using a well designed, validated diffusion cell system.
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- Authors: Smith, Eric W
- Date: 1988
- Subjects: Transdermal medication Skin absorption Dermatologic agents
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3815 , http://hdl.handle.net/10962/d1004741
- Description: The current world-wide interest in transdermal drug delivery makes the prospect of valid in vitro diffusion cell methodology highly attractive. A new laboratory diffusion cell has been designed and constructed based on theoretical principles and practical permeation reports surveyed in recent literature, and has been applied to the monitoring of betamethasone 17-valerate permeation. The cell performance has been validated with respect to hydrodynamic mixing efficiency and temperature of the receptor phase. The steady-state permeation of this corticosteroid has been monitored through various synthetic and animal membranes in order to select the most appropriate media for in vitro study. The permeation of betamethasone 17-valerate has been monitored from various types of commercial and extemporaneously prepared semisolid topical formulation (cream, lotion, ointment and scalp application), through silicone membrane, human and weanling pig stratum corneum, and full thickness hairless mouse skin, and these in vitro results have been compared to data from in vivo blanching assays, using the same formulations, in an attempt to correlate the findings. This experimental methodology has necessitated the development of ancillary analytical techniques. A column-switching high-performance liquid chromatographic method has been developed for the rapid on-line clean-up and analysis of betamethasone 17-valerate contained in the various topical formulations, which minimizes sample handling and extraction procedures. The method has been modified for the analysis of this corticosteroid in the isopropyl myristate receptor phase used in the in vitro permeation experiments, and scintillation counting of tritium-labelled water has been used to verify the integrity of the animal membranes. The comparison of in vitro permeation and in vivo blanching results indicate good correlation of the data in certain instances. The closest correlations have been observed when the human stratum corneum has been used in vitro and these results are compared to data from the occluded mode of the blanching assay. The results of the porcine and murine media have also correlated with the human in vivo data, whereas the silicone membrane appears applicable only in certain in vitro experiments. The results indicate that valid, comparative percutaneous absorption data may be obtained in vitro by using a well designed, validated diffusion cell system.
- Full Text: