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.
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- Date Issued: 1992
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.
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- Date Issued: 1992
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.
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- Date Issued: 1992
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.
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- Date Issued: 1992
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.
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- Date Issued: 1992
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.
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- Date Issued: 1992
The evaluation of indomethacin and theophylline oral controlled/modified-release dosage forms in vitro-in vivo correlations
- 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.
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- Date Issued: 1992