• The synthesis and pharmacological activity of 3-arylecgonine methyl ester and 3-carbamoyloxyecgonine methyl ester analogues: Probes for the characterization of the dopaminergic cocaine receptor

      Kline, Richard Harry, Jr.; Wright, Jeremy, Ph.D. (1991)
      Several analogues of 3-arylecgonine methyl ester were designed, synthesized and characterized by {dollar}\sp1{dollar}H and {dollar}\sp{lcub}13{rcub}{dollar}C NMR, IR and MS. The compounds were synthesized as racemates from 2,4,6-cycloheptatriene carboxylic acid or as enantiomerically pure compounds from 1 R-cocaine hydrochloride. These analogues were assessed for their ability to inhibit ({dollar}\sp3{dollar}H) cocaine binding to bovine striatal tissue and ({dollar}\sp3{dollar}H) dopamine uptake into striatal synaptosomes. Methyl(1RS-2-exo-3-exo)-8-methyl-3-phenyl-8-azabicyclo (3.2.1) octane-2 carboxylate was the most potent showing IC{dollar}\sb{lcub}50{rcub}{dollar} values for inhibition of ({dollar}\sp3{dollar}H) cocaine binding and ({dollar}\sp3{dollar}H) dopamine uptake of 22 and 133 nM, respectively. The racemates and the 1R-isomers proved to be equally potent inhibitors of binding and uptake. The 1RS-{dollar}\alpha{dollar}-dinitrophenylecgonine methyl ester analogue had the lowest potency. IC{dollar}\sb{lcub}50{rcub}{dollar} values for inhibition of binding and uptake were 11 and 30 {dollar}\mu{dollar}M, respectively. One of these compounds, 1R-{dollar}\beta{dollar}-(p-aminophenyl)ecgonine methyl ester, was designed and synthesized as a potential ligand for affinity chromatography. Several (1{dollar}R{dollar}-2-{dollar}exo{dollar}-3-{dollar}exo{dollar})-3-({dollar}N\sp\prime{dollar}-phenylcarbamoyloxy)ecgonine methyl ester analogues were also designed, synthesized, and spectroscopically characterized. Many of these compounds were synthesized as 1{dollar}R{dollar}-stereoisomers from 1{dollar}R{dollar}-ecgonine methyl ester in good yields. These compounds were tested for their ability to inhibit ({dollar}\sp3{dollar}H) cocaine binding to rat striatal tissue and ({dollar}\sp3{dollar}H) dopamine uptake into synaptosomes prepared from the same tissue. The most potent of these analogues was (1{dollar}R{dollar}-2-{dollar}exo{dollar}-3-{dollar}exo{dollar})-3-({dollar}N\sp\prime{dollar}-3{dollar}\sp\prime {dollar}-nitrophenylcarbamoyloxy)-8-methyl-8-azabicyclo (3.2.1) octane-2-carboxylic acid methyl ester. IC{dollar}\sb{lcub}50{rcub}{dollar} values for inhibition binding and uptake were 37 and 178 nM, respectively. In general, compounds which contained meta substituents on the phenyl ring of the C{dollar}\sb3{dollar} phenylcarbamate side chain were more potent than those with para substituents. Also, analogues which contained electron withdrawing groups on the same phenyl ring had considerably higher potencies than those with electron donating groups. Meta and para substituted amino 3-carbamoyloxyecgonine methyl ester analogues were found to be potential affinity chromatography ligands. Two isothiocyanato derivatives proved to bind irreversibly to the receptor, and two azido compounds were shown to function as photoaffinity ligands. These probes may be instrumental in the eventual isolation and purification of the cocaine receptor/dopamine transporter.
    • Partial purification and characterization of a C-methyltransferase from streptonigrin-producing Streptomyces flocculus

      Fox, Bonnie Marie; Speedie, Marilyn K. (1991)
      A C-methyltransferase which catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the C-3 position of the aliphatic side chain of L-tryptophan resulting in formation of {dollar}\beta{dollar}-methyl tryptophan, has been isolated from streptonigrin-producing Streptomyces flocculus. The enzyme catalyzes the first step in streptonigrin biosynthesis and is postulated to have a regulatory role in the pathway. The enzyme has been purified 217-fold by ammonium sulfate fractionation, followed by sequential gel filtration through Sephadex G-150 and Sephadex G-100 SF columns. Attempts at further purification have been hindered by very active proteases which co-purify with the enzyme. Protease inhibitors PMSF, pepstatin A, leupeptin, and trypsin inhibitor have failed to inactivate the protease activity. Based on comparison to reference proteins, the C-methyltransferase was estimated to have a molecular weight of 40,000 by Sephadex G-150 gel filtration. A narrow pH optimum of 7.5-8.0 was determined for the enzyme. The Sephadex G-100 SF fraction was highly unstable, losing 90 {dollar}\pm{dollar} 6% of its activity after 12 hours at 4{dollar}\sp\circ{dollar}C. S-Adenosyl-L-methionine and L-cysteine have been found to stabilize activity in the purified fractions. The enzyme is inhibited by sulfhydryl binding reagents, but no such inhibition is observed in the presence of substrate, suggesting an essential {dollar}-{dollar}SH group at or near the active site. Inhibition by carbonyl reagents was exhibited by the C-methyltransferase. Tritiated sodium cyanoborohydride treatment of the Sephadex G-100 SF fraction resulted in tritium incorporation and a concomitant 36 {dollar}\pm{dollar} 1% inactivation of the enzyme. These combined data led to the hypothesis that pyridoxal-5{dollar}\sp\prime{dollar}-phosphate may be involved as a cofactor in the C-methyltransferase. An enzymatic mechanism is proposed, and several studies related to this mechanism are presented.
    • Rational design of controlled release matrix tablets using an ethylcellulose dispersion and fluid bed granulation technology

      Smith, Bruce Philip; Hollenbeck, R. Gary (1991)
      Application of an aqueous polymeric dispersion to control drug release from matrix tablets was investigated. The polymeric dispersion of ethylcellulose was applied as a binder in matrix tablet formulations prepared by fluid bed granulation. Examined were the effect of excipients (dicalcium phosphate, lactose, and microcrystalline cellulose), drug solubility (chlorpheniramine maleate and hydrochlorothiazide), level of water insoluble binder (5 to 30% w/w), and compression force (300 and 600 kg) on the granule and resulting tablet characteristics. These objectives were approached through the use of mixture experimental design. Examined also were the physico-chemical properties of the granules and tablets to further explain the in vitro release behavior of the different formulations. The mean particle size ranged from 85 to 700 {dollar}\mu{dollar}m and the majority of tablets possessed tensile strength values which ranged from 5 to 25 kg/cm{dollar}\sp2{dollar}. A critical assessment of the theoretical framework was tested by observing how the solubility of drug and excipient(s) changed the consolidation characteristics, porosity, and tortuosity of the matrix. Compression of the fluid bed granules into tablets was a requisite for sustained release of drug since the granules themselves rapidly released the drug. Excipients and level of binder had a major impact on the release characteristics of the tablets. In general, for tablets containing chlorpheniramine maleate higher binder levels and compression force decreased the release of drug from the matrix. In contrast, for tablets containing hydrochlorothiazide higher binder levels decreased the release of drug but the release was insensitive to compression force. Drug solubility played a disproportional role in the release rate with the more water soluble drug producing the fastest release in part due to the disruption of the matrix infrastructure. Addition of the polymeric dispersion to the raw materials, was found to decrease the mean yield value by 17 to 72% indicating increased plasticity of the granulations. The database generated from the mixture design was used to rationally fabricate matrix tablets which had a specified release rate least sensitive to compression force. This study demonstrates that a controlled release matrix system can be produced using an aqueous ethylcellulose dispersion and fluid bed granulation technology.
    • Cholinergic pharmacology of tetrahydroaminoacridine (THA or Tacrine(registered trademark))

      Kiefer-Day, Jennifer Sue; El-Fakahany, Esam E. (1991)
      The acetylcholinesterase inhibitor tetrahydroaminoacridine (THA or Tacrine{dollar}\sp{lcub}\rm TM{rcub}{dollar}) is reported to enhance cognition in animals and some humans. As there is substantial evidence to support the involvement of the cholinergic system in memory, we investigated the effects of THA on muscarinic cholinergic receptors and their associated second messenger systems. Acute in vitro studies demonstrated that THA had the capacity to block muscarinic receptor mediated phosphoinositide hydrolysis and inhibition of cyclic AMP formation. Saturation binding studies revealed that the compound inhibited radioligand binding to muscarinic receptors through a mixed competitive/noncompetitive interaction. THA displayed a rank order of potency of M{dollar}\sb2{dollar} {dollar}>{dollar} M{dollar}\sb1{dollar} {dollar}>{dollar} M{dollar}\sb3{dollar}, but was a poor discriminator between muscarinic receptor subtypes. As demonstrated by steep displacement curves, however, marked changes in receptor occupancy may occur within a relatively narrow dose range. Thus, the slight selectivity of THA for the M{dollar}\sb2{dollar} receptor, which has been proposed to negatively regulate acetylcholine release, could complement its anticholinesterase properties to further augment cholinergic neurotransmission. Alternatively, concurrent blockade of postsynaptic receptors (whose function is essential for normal cognition) would oppose the beneficial effects of cholinesterase inhibition or presynaptic receptor blockade. Long term studies were also conducted as THA would normally be taken on a chronic basis. While THA (0.3-3 mg/kg) improves performance in animal cognition tests, a loss of efficacy and overt side effects occur at high doses. Furthermore, high dose THA treatment induces muscarinic receptor down-regulation over time. In contrast, we found that low dose THA treatment (0.3-3 mg/kg administered to mice for up to 32 days) did not alter the number of brain muscarinic receptors or the phosphoinositide response to muscarinic receptor agonists. Moreover, brain levels of THA were sufficient to inhibit 78-80% of acetylcholinesterase activity, regardless of treatment duration as measured 20 min after a 3 mg/kg dose. Thus, at a therapeutically relevant dose, THA inhibited the activity of brain acetylcholinesterase substantially, but did not affect the density of muscarinic receptors or their ability to activate second messenger systems. We conclude that the inconsistent reports of the efficacy of THA as a memory enhancer are unrelated to muscarinic receptor down-regulation, but may be related to receptor blockade.
    • The role of ecgonine methyl ester in the interpretation of cocaine concentrations in postmortem blood.

      Isenschmid, Daniel Stephan; Caplan, Yale H. (1991)
      The metabolism and hydrolysis of cocaine (COC) was studied in vitro, in vivo and postmortem to determine if measuring concentrations of the COC metabolites, benzoyl-ecgonine (BE) and ecgonine methyl ester (EME) would facilitate the differential diagnosis of COC related Medical Examiner cases. In vitro, in unpreserved whole blood, COC hydrolyzed to EME. In blood preserved by a pseudocholinesterase inhibitor, a solution of acetylcholinesterase, and phosphate buffer controls, COC hydrolyzed to BE. COC incubated in a solution of pseudocholinesterase and carboxylesterase hydrolyzed to EME. The rate of hydrolysis of COC increased as the pH and temperature of the specimen increased. COC was stable in blood adjusted to pH 5 and preserved with sodium fluoride or organophosphates for at least 150 days at 4C or lower. An in vivo study in human subjects given intravenous and/or smoked COC confirmed that BE is the principal metabolite of COC in blood. All COC was accounted for by BE. EME, when present, did not exceed 5% of the BE concentration.;EME arises in postmortem blood mainly as a result of non-metabolic hydrolysis of COC. Thus, in unpreserved blood specimens, the EME concentration can be added to the COC concentration to estimate the blood COC concentration prior to in vitro hydrolysis, while BE would be attributable to prior COC metabolism. Application of this theory to a study of Medical Examiner cases supported this conclusion. COC in blood was determined between 1 and 8 days following death and again 10 to 70 days after further storage (N = 10). The COC lost was accounted for by the EME formed. Good correlation (r = 0.9677) was observed when the blood COC concentrations in Medical Examiner cases were compared to blood COC concentrations predicted by the addition of blood COC and EME concentrations; hence, analysis for EME and estimation of perimortem COC concentrations can assist in defining deaths associated with COC use.
    • The characterization of heterogeneous surfaces using modified immersional calorimetry

      Demarest, Dudley Alvin, Jr.; Hollenbeck, R. Gary (1991)
      The present work describes the development and validation of a sensitive modified immersional calorimeter for assessing the integral interfacial energetics and wettability of tablet surfaces by aqueous dispersions of hydroxypropyl methylcellulose and its usefulness in predicting film coating results. Tablets manufactured from microcrystalline cellulose, dibasic calcium phosphate, lactose, and magnesium carbonate gelatin granulations were analyzed. Tablet surfaces were modified by varying the tablet porosity (by changing compression force) and specific surface energy (by adding various levels of magnesium stearate). A thermal gravimetric technique utilizing first derivative plots of drying curves of coating droplets on tablets as well as a kinematic assessment of tablet wettability are also described. Coating of tablets manufactured from the same excipient was performed in a 30 cm diameter Hi-Coater{dollar}\sp{lcub}\rm R{rcub}{dollar} side-vented coating apparatus using a competitive coating operation. The coating parameters were set to a level in which overwetting occurred thus "stressing" the coatability of the tablets. Tablet coating success was assessed by visual analysis, diametrical hardness increases, and with a size exclusion chromatographic (SEC) technique. This technique allowed the amount of coat applied to individual tablets to be determined at coat amounts of less than 1 mg. Immersional results were directly related to the surface area of interaction which depended not only on the porosity and penetration of the coating dispersion but, with low energy tablets, on the relative surface area of the immediate tablet surface. It was hypothesized that an immersional response "window" exists within which tablets will ultimately coat well. Those with larger immersional results will show rapid wetting of the tablet by the coating droplets which will dehydrate the droplets too rapidly for the coating to coalesce, and those with low responses will not allow enough spreading of the droplets to allow adequate coalescence. The comparison of the immersional results with the results of the tablet coating operation to some extent support such an hypothesis although the measurement of the surface area of interaction is essential in estimating the true surface energies of the interface from immersional results.
    • A study of selected physico-mechanical aspects of the extrusion and spheronization processes and their relationship to the design of pelletted pharmaceutical formulations

      Shah, Rajen Dhirubhai; Augsburger, Larry L. (1991)
      Pellets are becoming increasingly popular as unique delivery systems. Of the several processes available to prepare these pellets, extrusion/spheronization is better suited to produce pellets with the desired attributes such as narrow size distribution, high bulk density and low friability. The prevalent empirical approach to optimize this process warrants a complete understanding of its rheological requirements. The specific objectives are to identify and define criteria such as the plastic yield value which may govern the success of this process, and to assess the contribution of Microcrystalline cellulose (MCC) to this process. Toward this end, a twin screw extruder EXDS-60 was specially instrumented to measure screen pressure and screen temperature. In addition, techniques were developed to measure three properties of the wet mass, viz., yield value, tensile strength, and yield loci, in an attempt to relate to the qualities of ideal extrudates for spheronization. Using a model lactose/MCC system, statistically designed experiments were planned to study the relationship between formulation variables, rheological parameters of the wet mass, and quality parameters of the dried pellets. The instrumentation output provided new insights into the interplay between formulation and extrusion process variables and is further expected to assist in the design of formulations by providing objective measures of extrudability. The rheogram of the wet masses provided an important parameter in yield value. The tensile strength measurement with a specially designed split-die system provided valuable information regarding the mechanism of bonding within the wet mass. Also, the combination of yield value and weight of extrudate (as an indirect measure of tensile strength) could be used to predict the shape of the pellets. The yield loci parameters were useful in detecting over-wet granulations. Upon careful analysis of the experimental data, it was apparent that there was a critical range of rheological parameters within which pellets having optimum sphericity and narrow size distribution can be prepared. This critical range was defined as the rheological "window" within which both extrusion and spheronization can be carried out satisfactorily. Initially, MCC is perceived to be an essential "cure-all" ingredient for all extrusion/spheronization problems. Based on a consideration of the physico-chemical properties of MCC, and evidence from scanning electron microscopy, its unique contribution was attributed to its high internal porosity, and the formation of a particle network in the localized areas of the wet mass. In conclusion, the knowledge gained from this investigation facilitates the design of pelletted formulations by reducing reliance on empiricism.
    • The influence of aging on brain muscarinic receptors in the rat

      Surichamorn, Wanida; El-Fakahany, Esam E. (1991)
      Cognitive dysfunction is a dominant symptom that is generally reported in elderly as well as in Alzheimer's disease patients. The decline of such function is not recognized only in human beings but also in non-human mammalian species such as mice, rats, and monkeys. To explain this abnormality, several neurotransmitter systems have been demonstrated to decline accompanying the aging process and also they are postulated to play an important roles in the memory process, such as cholinergic, dopaminergic and noradrenergic system. However, a large number lines of evidence supported that the central cholinergic system especially in basal forebrain plays a pivotal role in memory function. These studies were designed to answer questions related to the effects of aging on the muscarinic receptor levels as well as its responsiveness upon the receptor stimulation. Fisher 344 rats were utilized as the study model since a large number of behavioral lines of evidence reported that the aged animals show a memory impairment similar to those in old humans. In this study, it is clear that there are age-associated specific alterations in the density of cell surface muscarinic receptors rather than the total receptors without changing their affinities in certain brain areas such as striatum and cerebral cortex. However, the alterations in receptor number is not associated with changes in the proportion as well as the affinities of pirenzepine-high affinity (M{dollar}\sb1{dollar}) and -low affinity (M{dollar}\sb2{dollar}: old nomenclature) in striatum, hippocampus and cerebral cortex with advancing age. In addition, the allosteric sites which are modulated by gallamine are not disturbed during the aging process. Furthermore, age-related changes in two main second messenger systems of muscarinic receptors such as the inhibition of adenylate cyclase (which may be linked to acetylcholine release) and PI hydrolysis (thought to play a pivotal role in the memory process) were studied. There were no age-related changes in the inhibition of Forskolin-stimulated cyclic AMP formation which contrasted to the well documented decline in acetylcholine release during aging, suggesting that these two responses are not linked to each other. Moreover, the data from the studies indicated that PI hydrolysis mediated by brain muscarinic receptors is not sensitive to age-induced changes in brain function. In addition, such receptor function was equally sensitive to blockade by phorbol esters and tetrodotoxin in young and aged rats. However, subtle changes might occur in discrete brain areas or in individual inositol phosphate species, especially inositol 1,4,5-trisphosphate, which cannot be detected by the methods employed in the present studies. It is also interesting to note that there are no age-related changes in the muscarinic receptor plasticity upon agonist pre-exposure, suggesting that treatment of memory deficit in the elderly with cholinermimetics should be performed with caution.
    • The qualitative and quantitative assessment of aldoxime stability by thermospray mass spectrometry

      Jakubowski, Edward Michael, Jr.; Callery, Patrick S. (1991)
      The application of thermospray mass spectrometry to qualitative and quantitative chemical degradation studies was assessed. A cholinesterase reactivator drug, 2-pralidoxime (2-hydroxyiminomethyl-1-methylpyridinium chloride, 2-PAM), was elevated as a model compound. Extrapolation of the reported first order rate equations for 2-PAM degradation in solution to thermospray conditions predicted the percent breakdown that was observed in the thermospray system. Both apparent gas-phase and apparent solution-phase reactions were observed and the activation energies calculated from thermospray data were in close agreement with available literature values. Breakdown of 2-PAM was dependent on solution pH, temperature, and flow rate. This evidence supported a strong contribution from liquid-phase reactions. A thermal flow reactor, modeled after the thermospray probe, was developed. The reactor consisted of a directly heated, 30 cm long stainless steel tube (0.015 cm id) with a thermocouple to monitor temperature changes. The tube was electrically insulated from the rest of the liquid chromatography-mass spectrometry system by using non-conductive polymer HPLC tubing. Either a cation exchange column or a C{dollar}\sb{lcub}18{rcub}{dollar} column in series with the reactor was used to separate possible degradation products of 2-PAM. The characteristic conversion of 2-PAM to the corresponding 2-pyridone (in equilibrium with 2-hydroxy-1-methylpyridinium ion) was related to temperature and flow rate. Also shown was the incorporation of {dollar}\sp{lcub}18{rcub}{dollar}O-water into 2-pyridone as a function of reactor temperature. The {dollar}\sp{lcub}18{rcub}{dollar}O studies supported the accepted mechanism of 2-pyridone production from 2-PAM. Incorporation of {dollar}\sp{lcub}18{rcub}{dollar}O into fragments of other aldoximes was demonstrated under thermospray conditions. In general, the reactor demonstrated that solution-phase degradation can occur under simulated thermospray probe conditions. The reactor can also be used with a UV detector to analyze drug stability at elevated temperatures.
    • Zero-order release through nonuniform drug distribution in a noneroding diffusional matrix: Theoretical design and actual manufacture

      Scott, Douglas Craig; Hollenbeck, R. Gary (1991)
      The theoretical design and actual manufacture of a non-eroding matrix pellet dosage form was undertaken. Non-uniform drug distribution in a non-eroding matrix can be used as a technique to achieve zero-order release. Flat slab and spherical geometry were examined through the development of a mathematical model to verify that it is theoretically possible to achieve zero-order release. Traditional non-eroding matrices, which contain drug uniformly distributed, do not yield zero-order release due to a constantly increasing barrier thickness which develops as drug diffuses from the matrix. Non-uniform drug distribution in a porous non-eroding matrix, in which drug is more concentrated in deeper matrix layers, creates an increasing depletion zone porosity as drug is leached from the matrix. This increasing porosity compensates for the increasing barrier thickness to achieve constant rate release. A common pelletization technique was employed to manufacture pellets. Previous investigations of this approach are few and have never combined a theoretical approach with a practical manufacturing process. An automated modified suspension layering technique was developed to create pellets containing drug non-uniformly distributed. A gradient pumping system used in conjunction with a specially fabricated Wurster column was used to produce these pellets. To examine the feasibility of this process, an investigation of the behavior of a water soluble drug, chlorpheniramine maleate, and a water insoluble drug, haloperidol, was undertaken. The model drugs and non-eroding matrices were characterized and actual release profiles were compared to theoretical profiles. In the case of the water soluble drug, an alternate release mechanism appears to predominate and the system does not behave as predicted by theory. In the case of the water insoluble drug the model fits well. A slight burst of drug is observed and is due to an initial porosity which exists in the matrix. The differing performance of these drugs appears to be due to their physico-chemical character. The water insoluble drug conforms to the assumptions and conditions of the model and demonstrates that non-uniform drug distribution in theory and actuality can be used to achieve zero-order release.
    • Kappa-opioid induced regulation of mu-opioid mediated effects on EEG power spectra and behavior in rats

      Paquette, Nicole Corinne; Young, Gerald A. (1991)
      The regulatory effects of kappa-opioid ligands on mu-opioid induced changes in EEG power spectra and behavior were evaluated in rats receiving various acute and chronic treatments. Rats were implanted with cortical EEG electrodes and i.c.v. and/or intravenous (i.v.) cannulae. EEG spectral parameters were derived from digitized EEG samples with spectral analysis techniques. Pretreatment with intracerebroventricular (i.c.v.) injection of dynorphin, morphine and dynorphin/morphine resulted in quantitative and qualitative changes in EEG power spectra in rats given i.c.v. morphine 24 hr later. I.c.v. injections of morphine (20 {dollar}\mu{dollar}g/rat) produced high-voltage, slow-wave EEG bursts (1-10 Hz). Injections of i.c.v. morphine in rats pretreated with i.c.v. dynorphin (20 {dollar}\mu{dollar}g/rat), morphine (20 {dollar}\mu{dollar}g/rat) or dynorphin/morphine twenty-four hours earlier, produced quantitative increases in absolute EEG spectral power. Injections of i.c.v. morphine in rats pretreated with i.c.v. dynorphin/morphine 24 hr earlier, also produced qualitatively different EEG power spectra with a predominant peak in the 4-6 Hz band, similar to the EEG power spectra seen after acute administration of kappa opioids. Correlated changes in sensitivity to antagonism of these EEG effects by naloxone were also found. Thus, Dynorphin may act as a possible regulator of certain {dollar}\mu{dollar}-opioid receptor-associated phenomena, such as morphine-induced EEG bursts at a binding site which is nor-BNI insensitive. Chronic administration of morphine, a {dollar}\mu{dollar}-opioid selective agonist, for seven days resulted in tolerance development to EEG bursts and EEG absolute power, but not to latency to onset of slow-wave sleep (SWS). Chronic morphine administration was also associated with increases in the duration of hyperexcitability. Chronic administration of U-50,488H, a selective {dollar}\kappa{dollar}-agonist, was not associated with any changes in the duration of EEG bursts, latency to onset of SWS or duration of sedation. The coadministration of U-50,488H and morphine produced no significant tolerance development to the duration of EEG bursts and latency to onset of sleep, but increased the duration of sedation. Morphine or ethylketocyclazocine (EKC) challenges before and after chronic treatment with either morphine or U-50,488H resulted in greater tolerance development to the duration of EEG bursts and to increases in EEG absolute spectral power compared to the group receiving chronic coadministration. (Abstract shortened with permission of author.)
    • Photosensitization by diaziquone: Correlation between diaziquone cytotoxicity and photoinduced free radicals in MCF-7 cells

      Al-Nabulsi, Isaf; Wright, Jeremy, Ph.D. (1991)
      The ability of visible light to enhance the activity of diaziquone (AZQ) was evaluated in MCF-7 human breast cancer cells. Exponentially growing monolayers of MCF-7 cells were incubated for 1 hr with AZQ (IC{dollar}\sb{lcub}90{rcub}{dollar}, 0.05 {dollar}\mu{dollar}M, IC{dollar}\sb{lcub}50{rcub}{dollar}, 0.3 {dollar}\mu{dollar}M, or various concentrations of AZQ) prior to variable time intervals of visible light irradiation. Irradiations were performed using a 100W quartz-halogen lamp or 100W mercury arc lamp with a dose rate of 30 or 170 mW/m{dollar}\sp2{dollar}, respectively. The effect of visible light and/or AZQ on cellular growth was determined by clonogenic assay. The results show that MCF-7 cells were sensitive to growth inhibition by AZQ. Without AZQ, visible light irradiation had no effect on cell survival, while with AZQ, visible light potentiated its cytotoxicity by a factor of 1.6 at 10% survival. This potentiation of AZQ activity is correlated with the formation of free radicals (hydroxyl radicals and AZQ semiquinone) and with the production of DNA strand breaks as measured by electron paramagnetic resonance and gel electrophoresis, respectively. These results support the hypothesis that free radical formation is part of the mechanism of action of AZQ. Moreover, they indicate that visible light irradiation can increase the activity of AZQ and may allow its use in the treatment of tumor in human patients.
    • Evaluation of a capacitive sensor for wet granulation monitoring in a high shear mixer

      Corvari, Vincent John; Augsburger, Larry L. (1992)
      The objectives of this dissertation are to evaluate and compare a new capacitive sensor, a watt meter and a strain gaged torque sensor for wet granulation monitoring in a high shear mixer. Toward this end, a 10 L vertical high shear mixer was instrumented to monitor power consumption, torque, moisture distribution and capacitive changes during agglomeration. The percent moisture content related linearly to the amplitude channel response. Yield values provided a rheological property to relate with power consumption and torque measurement. In a comparative study of the two most commonly used monitoring techniques, torque measurement was shown to be more sensitive than power consumption measurement, but this increased sensitivity did not offer any advantage over power consumption measurement. In another comparative study the amplitude channel of the capacitive sensor appears to more clearly differentiate between binder levels in hydrous lactose-hydroxypropyl methylcellulose granulations than either power consumption or torque measurement, based on particle size distributions. Four formulations were used to evaluate the effect of varying agitation rate and rate of fluid addition on the granulation endpoint determined by the capacitive sensor. For hydrous lactose (Lactose) with 4% polyvinylpyrrolidone K 29/32 (PVP), dicalcium phosphate anhydrous milled (DCP) with 5% PVP and a one to one mixture of Lactose and microcrystalline cellulose (MCC 102) with 5% PVP, the amplitude channel of the capacitive sensor predicted similar endpoints based on particle size, flow rate and crushing strength-compression force profiles under varying agitation and addition rates. The apparent independence of the amplitude channel to variations in agitation rate and rate of liquid addition suggests that this component of the capacitive sensor may be scaled up. The amplitude channel was successfully scaled up from a 50 L to a 130 L horizontal high shear mixer based on particle size, flow and crushing strength-compression force profiles for Lactose/PVP formulations. In addition, the amplitude channel was able to compensate for differences in moisture distribution when the chopper was employed in the scale-up study. A centrifugation technique, thermogravimetric analysis and nuclear magnetic resonance (NMR) inversion recovery technique were developed to compare the interaction of moisture with each of the filler systems used in the endpoint detection study. (Abstract shortened by UMI.)
    • Pharmacological treatment during status epilepticus: Metabolic and electrographic studies

      Jones, Bruce Edward; Buterbaugh, Gary G. (1992)
      The present study examined the effect of diazepam, pentobarbital, THIP (4,5,6,7-tetrahydroisoxazolo-(4,5c)-pyridone-3-d)), valproic acid and MK-801 treatment on metabolic, electrographic and behavioral components of a pilocarpine facilitated model of status epilepticus (pfSE). This study demonstrated that amygdala kindled rats pretreated with a non-convulsive dose of pilocarpine and electrically stimulated produce a stable seizure characterized by high-amplitude, fast-frequency EEG and increased ({dollar}\sp{lcub}14{rcub}{dollar}C) -2-deoxy-D-glucose ( ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG) uptake. Additionally, the present study demonstrated diazepam treatment during pfSE resulted in a dose-dependent decreased ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake. The somatosensory cortex appeared most sensitive whereas the amygdala, entorhinal cortex, reuniens nucleus, lateral septum and claustrum-endopiriform exhibited a resistance to diazepam with respect to ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake. The effect of diazepam was attenuated by the selective benzodiazepine antagonist Ro-15-1788. As with diazepam pentobarbital reduced regional ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake, however, the effect was not dose-dependent. THIP, valproic acid, and MK-801 did not alter the regional ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake pattern during pfSE. Except for pentobarbital no compound administered resulted in alterations in EEG. Therefore diazepam treatment was the only treatment which resulted in reductions in ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake without attenuation of EEG. Furthermore the data did not establish a link between potentiation of GABA receptor-mediated inhibition of neuronal activity and the effect of diazepam. However, there was evidence to suggest that kindling is important to the effect. This study also provides evidence that increased ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake was associated with high dose pilocarpine-induced SE. However, these data did not indicate that diazepam treatment resulted in reduced ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake in this particular model. In summary these data clearly demonstrate a dissociation of ({dollar}\sp{lcub}14{rcub}{dollar}C) -2DG uptake and electrical activity as a result of diazepam treatment. Furthermore these data suggest that diazepam mediates this effect through a specific receptor-mediated effect, however, no connection was established between the effect of diazepam and potentiation of GABA receptor-mediated inhibition of neuronal activity but it does appear as if kindling is necessary.
    • Computer-assisted analysis of structure-activity relationships of philanthotoxin analogues

      Huang, Yu-Ing; Callery, Patrick S.; Pou, Sovitj (1992)
      Discovery of several venom toxins from spiders and a digger wasp as potent glutamate receptor antagonists has provided new approaches for the investigation of glutamate receptors. Philanthotoxin-433, a wasp toxin and an allosteric inhibitor of nicotinic acetylcholine (nAch) receptors, and numerous analogues have been reported to be potent antagonists of quisqualate receptors. In this dissertation, molecular mechanics and semiempirical (AM1) calculations were performed on 28 philanthotoxin (PhTX) analogues. Conformational analyses were carried out by systematic/grid searches to determine energy minima. In the calculated preferred conformations, two intramolecular hydrogen bonds were found to stabilize the conformers; one hydrogen bond between the two amides and another between the aromatic ring {dollar}\pi{dollar}-cloud and the polyamine chain amino hydrogens. Antagonistic potencies against the quisqualate and nAch receptors were explained through a spatial occupancy model of the PhTX analogues. Quantitative correlations were determined by comparative molecular field analyses (CoMFA). These studies revealed that the quisqualate receptor antagonism observed for the PhTX analogues correlated well with their structural modifications at the butyryl, tyrosyl and polyamine moieties. No significant correlation between the nAch receptor antagonism and PhTX structures was found. A CoMFA model for quisqualate receptor antagonism was established and had a correlation coefficient of 0.898 and a predictive r{dollar}\sp2{dollar} of 0.614. The logarithm of the IC{dollar}\sb{lcub}50{rcub}{dollar} values provided the best descriptor for the biological data in these CoMFA studies. The relative contribution of steric and electrostatic fields to the CoMFA model was 64% and 36%, respectively. A spermine chain with a free terminal amino functionality was found to be optimal for quisqualate receptor antagonism while the tyrosyl and butyryl moieties were predicted to be better regions for structural modifications leading to new antagonists.
    • Elucidation of the nature of the plug formation process and its implications in a dosing disk machine using an instrumented Hofliger Karg GKF 330 capsule filling machine

      Cropp, John Worth; Augsburger, Larry L. (1992)
      Particle deformation, stress transmission, and bonding in compacts have been studied extensively at higher force levels such as those associated with tableting equipment. However, fundamental questions exist regarding the nature of particle behavior during compaction at low forces, such as seen with the dosator and dosing-disk type automatic capsule filling machines. There are published reports in the literature citing unusual dissolution behavior of finished capsule dosage forms for which no explanation is available. An Hofliger Karg GKF 330, a dosing-disk machine, previously instrumented to monitor tamping force, was further modified to allow for simultaneous monitoring of brass ring movement and movement of individual tamping pins. These data were used to determine powder displacement. Powder displacement data were combined with tamping force data to determine work of compaction and perform Heckel analysis. Model formulations, exhibiting classic brittle or plastically deforming type behavior, were evaluated using the instrumented Hofliger Karg GKF 330 by varying overload spring type, pin penetration setting, and number of tamps. Capsule plugs were examined for evidence of particle deformation, plug strength, plug weight, and release of a slightly soluble drug, hydrochlorothiazide. Plugs were evaluated using scanning electron microscopy, BET gas adsorption surface area, and mercury intrusion porosimetry to provide detailed information to the behavior of materials when compacted at low forces. Microcrystalline cellulose based plugs exhibited minimal particle deformation whereas anhydrous lactose based plugs exhibited evidence of fracture. Both formulations exhibited increased plug strength and weight with higher tamping forces which resulted from the use of a larger wire diameter overload spring, increased pin penetration setting, or both. No effect upon the release of hydrochlorothiazide was observed as a result of varying number of tamps or tamping force.
    • An examination of the theory of reasoned action and pharmacists' intention to provide medication counseling

      Siganga, Walter Wesonga; Beardsley, Robert S. (1992)
      Although it has been shown that counseling can benefit patients and pharmacists alike, it is well documented that pharmacists are reluctant to counsel patients about medications. This study was conducted to explain and predict pharmacist's counseling intentions and behavior using Fishbein and Ajzen's Theory of Reasoned Action (TRA), with the intention of explaining why pharmacists do not counsel as often as they could. The main goal of the study was to provide a better understanding of pharmacists' counseling behavior. A random sample of 317 Maryland pharmacists completed a questionnaire that measured their attitudes and subjective norms towards intention to provide counseling. Self-reports of behavior were collected through interviews from a sub-sample of 52 pharmacists. Three hypotheses dealing with pharmacists' attitudes and subjective norms towards their intention to counsel and the relationship between intention to counsel and their reported counseling behavior were developed and tested. Results indicated that pharmacists have positive attitudes and subjective norms towards counseling, that attitude was a better predictor of counseling than subjective norm and that demographic characteristics could influence pharmacists' counseling intentions. Reported counseling behavior through interviews differed from perceived counseling intentions. Overall, the results indicated that pharmacists have positive counseling behaviors. However, the results explained a small amount of variance. A new or modified version of the model depicting the Theory of Reasoned Action could be used to explain more of the behavior. The results have implications for designing interventions that may improve counseling and also affect medication costs. There are implications also for improving patient compliance, mandatory counseling, federal and private insurance programs. Practitioners and pharmacy students may benefit through continuing education programs that use the knowledge about attitudes and subjective norms. The patient counseling role of pharmacists may be stressed and pharmacists encouraged to counsel.
    • The evaluation of the metabolism of N-methylspiperone and its effect on kinetic and receptor binding parameter estimation

      Miller, Ann Kay; Young, David G. (1992)
      N-methylspiperone (NMSP) is a butyrophenone derivative that binds preferentially to dopamine D2 receptors in the brain of animals and man. This dissertation describes the development of a specific and sensitive HPLC assay for quantitation of NMSP in guinea pig plasma and brain. The metabolism of NMSP is studied in guinea pigs and rats after multiple intraperitoneal injections of NMSP. Three important metabolites found in guinea pig plasma and brain were identified as reduced NMSP, spiperone, and reduced spiperone. Only spiperone was found in rats. Furthermore, reduced NMSP was found to be a radiolabeled metabolite after single or multiple doses of {dollar}\sp3{dollar}H-NMSP and after a single dose of {dollar}\sp{lcub}11{rcub}{dollar}C-NMSP in guinea pigs. The plasma disposition of NMSP and {dollar}\sp3{dollar}H-NMSP in guinea pigs after a single intravenous dose was best described by a biexponential decline. The half-lives of NMSP distribution and elimination were 11.7 and 289 minutes, respectively. Reduced NMSP was found to follow a mono- or biexponential decline in plasma after a single intravenous dose. The mean elimination half-life was 54.7 minutes. The "donor-recipient" parameter estimation method was evaluated for usefulness in NMSP receptor binding studies. This method includes estimation of transfer rates of formation of a radiolabeled metabolite in the body and transfer of the metabolite into and out of the brain. Data was simulated for a radiolabeled parent compound and its radiolabeled metabolite in the plasma and in the two types of brain regions--those with and without specific binding of parent to the receptors. All parameters except the rate constant of metabolite elimination from the body were estimated within 1% of the true value. Parameters estimated by non-linear least squares regression analysis were found to be similar to those from the "donor-recipient" method. In evaluating the "donor-recipient" method when specific binding is assumed to be reversible, only K23 was poorly estimated. The "donor-recipient" method does provide accurate estimation of all parameters of interest in the receptor binding models determined in this analysis. However, K23, the rate of sequestration into the brain compartment, is only accurately estimated when it is truly represented by a first order process and K23 is zero. (Abstract shortened with permission of author.)
    • EEG, EEG power spectral and behavioral profiles of two inbred rat strains upon acute and chronic opiate exposure

      Mayo-Michelson, Lieser; Young, Gerald A. (1992)
      Utilizing EEG, EEG power spectra and behavioral parameters, two inbred rat strains, Lewis (LEW) and Fischer 344 (F344) were compared following acute morphine, chronic morphine and acute ethylketocyclazocine (EKC) treatment. Following acute morphine administration, the LEW rats exhibited greater duration of morphine-induced EEG slow-wave bursts and associated behavioral stupor. For both LEW and F344 rats, suppression of onset to slow-wave sleep increased in a dose-related manner. Regarding morphine dose, all spectral parameters differed except peak frequency. For the chronic morphine study, LEW and F344 inbred rat strains were exposed to morphine (iv) over a period of seven days in order to discern differences in tolerance development and physical dependence. Following morphine injection, the LEW group exhibited a greater mean total amount, as well as a greater rate of reduction, of stuporous behavior across the seven days tested. The LEW rats exhibited a positive linear profile of opiate-induced hyperexcitability, while a quadratic profile emerged for the F344 group. Over days, differences in patterns of latency to onset of slow-wave sleep between the two strains were also revealed; the F344 rats exhibited a greater change (as reduction of suppression) across the seven days tested. Naloxone was administered (iv) following the seven days of morphine treatment in order to delineate differences in dependence. The LEW animals reflected a greater amount of withdrawal-type behavioral responses, e.g., wet dog shakes, diarrhea, body stretch, sluggish behavior. However, assessment of power spectral parameters pre- and post-naloxone treatment revealed a greater withdrawal response for the F344 group. Acute administration of EKC also revealed differences between LEW and F344 inbred rat strains. EKC-induced EEG slow-wave bursts and associated behavioral stupor increased in a dose-related manner, and was greater in duration, for the LEW animals; a less robust, quadratic trend over doses was displayed by the F344 group. Separation of the phases revealed rat strain differences; overall, the LEW animals displayed a dose-related EEG response. Except for total power, the F344 group displayed little variation across the three doses tested in the burst phase; the interburst phase reflected dose-related differences for this group but to a lesser extent in comparison to the LEW rats. (Abstract shortened with permission of author.)
    • Incidence and outcomes of reported poisoning in the elderly

      Gondek, Kathleen; Lamy, Peter P. (1992)
      Both inadvertent and intentional poisonings are responsible for increased morbidity and mortality in the elderly. This investigation estimates the incidence and describes the basic epidemiology of drug-related poisonings among community-dwelling elderly in the United States. The American Association of Poison Control Center's database and the National Medical Expenditure Survey, respectively, were used to calculate numerator and denominator data necessary to estimate incidence. There were approximately 135,000 calls to poison control centers from those 60 years of age and over between 1986 and 1990. For each study year, incidence estimates were adjusted for changes in prescribing based on data from the National Disease and Therapeutic Index.;Household and cosmetic products account for 30 percent of all calls regarding non-drug poisonings and topical medications accounted for more than 7,600 calls to poison control centers across the country. Approximately 90 percent of topical drug poisonings were via ingestion and seven people died. The majority of calls regarding non-drug and drug poisonings were from females.;More detailed analyses for antidepressants and benzodiazepines were performed to test hypotheses and develop a model predictive of the risk of adverse outcomes. Statistically significant differences in outcome were found among benzodiazepines and antidepressants after controlling for age, sex and census region. Outcome is coded as no effect, minor, moderate, major and death. Logistic regression analyses models were used to predict poisoning outcome, the dependent variable, with the remaining independent variables in the dataset. The variable admission to a health care facility was seen as a proxy measure for outcome and warranted a separate analysis. Several variables in each analysis are significant; confidence intervals were calculated to determine the effect size. For example, the odds ratios indicate that the estimate of relative risk of admission to a health care facility for antidepressants is related to an intentional poisoning, being symptomatic, living in the Mid-Atlantic census division, an adverse drug reaction (at normal doses or normal use) and the number of substances ingested. Thus, the study provides health care workers with an increased knowledge of drug misuse in the elderly and the adverse consequences of such use.