Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Ondrej Tomáš Supervisor: PharmDr. Marie Vopršalová, CSc. Consultant supervisor: mjr. PharmDr. Vendula Hepnarová, Ph.D. Title of diploma thesis: Evaluation of newly prepared insecticides in vitro Malaria is a widespread infection and one of the most dangerous diseases transmitted by insects. It threatens lives of millions of people all around the world, thus its regulation is necessary. Most common malaria vectors are mosquitoes of genus Anopheles. Novel structures of insecticides with selective inhibition of mosquito acetylcholinesterase are subjects of research, with an intention to deal with this problem. The aim of this work was to test six newly prepared succinimide derivatives with insecticidal potential. The ability of these compounds to inhibit Anopheles gambiae mosquito (AgAChE) and human acetylcholinesterase (hAChE) was evaluated. Leading structures of these compounds were also tested to find relations between chemical structure and biological activity. The modified Ellman's method was used to obtain the half maximal inhibitory concentration (IC50) values for both enzymes. Tested substances were able to inhibit only hAChE and none of them displayed activity against AgAChE. Compound...

Experiments were performed to determine the degree of overlap in the distribution of muscarinic receptors and cholinergic innervation of the rat heart. Localization of muscarinic receptors was determined by autoradiography with [3H]quinuclidinyl benzilate. Adjacent sections were stained for acetylcholinesterase to determine innervation. The distribution of muscarinic receptors and cholinergic innervation overlapped in cardiac parasympathetic ganglia, nodal tissue, His bundle-Purkinje system, vena cava and pulmonary veins. Cholinergic innervation to the right atrium was greater than to the left atrium while muscarinic receptor density was equal in the two atria. Innervation of the ventricles was confined primarily to the base of the right ventricle. A low density of muscarinic receptors was observed throughout the ventricles. Neither cholinergic innervation nor muscarinic receptors were detected in the pulmonary trunk, ascending aorta or cardiac valves. Muscarinic receptors and cholinergic innervation in the nodal regions, ventricular conduction system and myocardium probably mediate negative chronotropic, dromotropic and inotropic effects of vagal nerve stimulation. Muscarinic receptors at sites not containing cholinergic innervation may be associated with noradrenergic nerves of the myocardium.

Adult female cats (2.4-2.8 kg) underwent surgical removal of the left ciliary ganglion under pentobarbital anesthesia. Twenty-one, 560 and 616 days later, pupil size of both left and right (control) eyes was measured in response to progressively increasing doses of carbachol applied topically. By 21 days after denervation, ganglionectomized eyes showed marked supersensitivity to the miotic effects of pilocarpine and carbachol. By 560 days, however, responsiveness of the denervated eyes to lower and intermediate doses of carbachol was the same as that of contralateral control eyes, while responsiveness to higher doses was significantly reduced. Responsiveness to both lower and higher doses of carbachol was significantly less than that of the controls on the 616th day. Ganglionectomized eyes showed no pupillary response to light 14, 562, or 620 days after surgery. Histochemical analysis of iris tissue collected from eyes of these cats 720 days after ganglion removal revealed an almost complete absence of acetylcholinesterase-containing nerve fibers on the denervated side. These findings indicate that the return to normal or lower sensitivity to carbachol of denervated eyes at prolonged periods after ciliary ganglion removal is not due to significant cholinergic reinnervation of the iris sphincter muscle.

The acetylcholinesterase (AChE) content of rat vagal efferent neurons was studied. Retrograde transport of horseradish peroxidase (HRP) by cut vagal axons provided a means for localizing efferent cell bodies; tissue sections were then processed for the simultaneous visualization of HRP and AChE. A dorsal vagal efferent column contained the dorsal motor nucleus of the vagus, as a primary component, and extended caudally into the upper cervical spinal cord. A ventral column contained neurons in the nucleus ambiguus and the surrounding reticular formation. Although most of the vagal efferent neurons stained with moderate to heave intensity for AChE there were some HRP-labeled cells that contained little AChE and a small percentage in which AChE was absent. In spite of the fact that AChE has been demonstrated in certain non-cholinergic neurons, it has also been found in all cholinergic neurons. Therefore, the presence of AChE has been regarded as a necessary (but not sufficient) component for identifying cholinergic neurons. The absence of AChE in a small percentage of the vagal efferent neurons indicates that some preganglionic parasympathetic fibers in the vagus nerve are not cholinergic.

The brainstem localization of acetylcholinesterase (AChE)-containing neurons projecting to the anteroventral thalamic nucleus (AVN) was studied in rats. The AVN is one of several forebrain regions innervated by the AChE-containing dorsal tegmental pathway described by Shute and Lewis. In the present study, horseradish peroxidase (HRP) was injected into the region of the AVN to determine the brainstem origin of afferent projections. Alternate sections of tissue were stained for HRP or AChE. HRP-labeled neurons were found in the laterodorsal tegmental nucleus (LTN) and the locus coeruleus. Examination of adjacent sections revealed AChE-containing neurons in both of these nuclear regions. Combined HRP/AChE histochemistry demonstrated that transported HRP and AChE were in the same cells. In further experiments, unilateral lesions of the LTN were found to cause a decrease in AChE staining of the ipsilateral AVN. Destruction of the locus coeruleus had no effect. In combination with available evidence, the present findings suggest that cholinergic neurons in the LTN innervate the AVN. © 1980, All rights reserved.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Soňa Knápková Supervisor: PharmDr. Daniela Hulcová, Ph.D. Title of diploma thesis: Amaryllidaceae alkaloids as model structures for the development of new potential drugs The family Amaryllidaceae includes a large number of genus of flowering plants as Hippeastrum, Narcissus or Zephyrantes. All of them contain specific Amaryllidaceae alkaloids, which are characteristic of this family due to their chemical structures. The discovery and study of these alkaloids has attracted the attention of many scientists due to the diverse biological activity of these compounds, such as cytotoxic, anticholinesterases, antibacterial and antiviral effects. Plants of the genus Hippeastrum have been used in traditional medicine to treat tumors and inflammatory disorders. This use can be explained by the alkaloids, which it contains. It is mainly lycorine, haemanthamine and pancristatine. These compounds have an antitumor effect. The species Hippeastrum cv. Ferrari is further rich in the alkaloid vittatine. In some research, the simple semisynthetic derivatives of haemanthamine displayed promising inhibitory activities against cholinesterases. For this reason, vittatine was chosen as next lead-structure, for preparation of...

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Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by catalyzing the hydrolysis of the neurotransmitter acetylcholine (ACh). Inhibition of AChE has proven an effective treatment for the memory loss exhibited by early stage Alzheimer's disease (AD) patients; four AChE inhibitors (AChEI) have been approved by the FDA for this purpose. The first AChEI approved for the palliative treatment of AD-related memory loss was 9-amino-1,2,3,4-tetrahydroacridine (tacrine). Inhibition of AChE may present either therapeutic or toxic effects depending upon the dose administered. With the goal of discovering safe and effective pesticides to control the population of Anopheles gambiae, a malaria-transmitting mosquito indigenous to Sub-Saharan Africa, the reoptimization of the tacrine pharmacophore was undertaken. Because the optimized drug would necessarily be a poor inhibitor for human AChE (hAChE), initial ligand design focused on modification to tacrine known to negatively impact the inhibition potency for hAChE. Ultimately, an AChEI was discovered, which exhibited micromolar inhibition of Anopheles gambiae AChE (AgAChE) and essentially no potency for hAChE. Two units of this lead compound were tethered through an alkyl chain to yield a nanomolar inhibitor of AgAChE that was more than 1,100-fold selective for the mosquito enzyme over hAChE. Dimerization of an active inhibitor is an effective strategy to increase the potency and selectivity of AChEI, and many examples of tacrine hetero- and homodimers complexed to AChE can be found in the RCSB Protein Data Bank (PDB). The bond formed between the exocyclic amine moiety and the heterocyclic ring system of tacrine is analogous to an amide bond when tacrine is protonated. Therefore, the rotational profile of protonated N-alkyltacrine should exhibit a conformational profile in which dihedral angles significantly out of the plane formed by the ring system are associated with high energies relative to those when the dihedral angles are nearly coplanar with the ring system. The barrier of rotation (ΔG^‡) produced by this phenomenon in two tacrine derivatives and two quinoline derivatives was experimentally determined using dynamic 1H NMR. These values were compared to density functional theory (DFT) derived values for the same phenomenon. Furthermore, since the ΔG^‡ proved to be impossible to experimentally determine for the optimal model compound for the active site portion of tacrine dimers, N-methyltacrine, the DFT method employed for modeling the ΔG^‡ of the tacrine and quinoline analogs was used to computationally derive the entire rotational conformation diagram of N-methyltacrine. The calculated values were then used to comment on the relative energies of adopting certain conformations found in the X-ray crystal structures of dimer/AChE complexes. / Ph. D.