Past and future of an IMI-PharmaTrain (IMI-PhT)-initiated multinational pharmaceutical medicine course at the Semmelweis University in Hungary.

The pharmaceutical medicine course at the Semmelweis University of Budapest, Hungary, was initiated as part of the Innovative Medicines Initiative (IMI is the main program, IMI-PharmaTrain is one of the IMI projects) Pharmaceutical Medicine Training Programs (16 IMI Call 2008/1/16). The aim was to extend training in the development of pharmaceutical medicine to those EU member states where no such education was present. The final program envisaged the development of a cooperative education supported by universities located in Central and Eastern Europe. It was considered to be the economically and scientifically most viable approach to combine the expertise from these countries to form a united teaching staff and provide education jointly for young professionals of the region. Semmelweis University was selected to manage this coordinated program. In this report, we describe the organization and functioning of this international university-based pharmaceutical medicine education project called the Cooperative European Medicines Development Course (CEMDC) and evaluate its successes and shortcomings. During the pandemic, the educational course was interrupted. The follow-on program is reorganized as a postgraduate MSc course named "Semmelweis Pharma MBA" and will be started in 2025. It will continue the established PharmaTrain educational tradition. However, it will deal in more detail with the transition from basic pharmacological to industrial research, as well as biopharmaceutical formulation and manufacturing and marketing aspects of medicines development.

Analysis of thymidine kinase activity and glucocorticoid-binding capacity in the thymuses of healthy and tumor-bearing chickens.

Thymidine kinase activity in the lymphoid organs of healthy and tumor-bearing chickens was studied after hydrocortisone or dexamethasone treatment. Glucocorticoid hormones (10 mg/100 g body wt) administered twice in 48 hours resulted in an 80% decrease in thymidine kinase activity in the thymuses of healthy chickens, whereas birds with transplantable MC 29 chicken hepatoma failed to respond to the hormones. In the bursae of Fabricius of healthy or tumor-bearing chickens, thymidine kinase activity did not change considerably after administration of hydrocortisone or dexamethasone. The steroid-binding capacity in cellfree systems prepared from thymuses of tumor-bearing birds was significantly decreased. Thus the difference in steroid-binding capacity between thymuses of healthy and tumor-bearing chickens correlated well with, and could account for, the failure of hydrocortisone and dexamethasone to reduce the thymidine kinase level in tumor-bearing birds.

Kinetics of the hormone-receptor interaction. Competition experiments with slowly equilibrating ligands.

Kinetics of formation of protein-tracer complex in the presence of competitor were calculated. The parameters were chosen so that they should realistically describe the in vitro association of steroid hormones with their receptors. Time necessary for equilibration depends on four rate constants in addition to initial concentrations and may be more than 1000 min. Competitors forming complexes with the protein that dissociate faster than the protein tracer complex have relative binding affinities apparently decreasing with incubation time. Conversely, relative binding affinity apparently increases with time if the protein-competitor complex dissociates more slowly than the protein-tracer complex. Moreover, lack of equilibration is not easily detected. It is suggested that kinetic analyses, more detailed than usual, precede competition experiments.

Kinetics of the glucocorticoid hormone-receptor interaction. False association constants determined in slowly equilibrating systems.

The usual way of in vitro determination of association constants for hormone-receptor complexes is criticized. It is shown that if incubation time is short, relative to the half-life of the hormone-receptor complex, the value of the apparent Ka is proportional to the time of incubation. No sign of lack of equilibrium is apparent from the Scatchard plots. The case of rapidly denaturing receptor molecule is also discussed, with similar conclusions. Although terminology and examples are taken from the field of the glucocorticoid receptor research, all deductions are valid for other systems with similar association (and denaturation or monomolecular transformation) mechanisms and kinetic parameters.

Characterization of the partially purified, ligand-free glucocorticoid receptor.

A new method was developed to synthesize a cortexolone-substituted affinity matrix, based on the fast, mild and quantitative reaction between alpha-ketomesylates and thiols. The resulting cortexolone-Sepharose absorbed easily the cytosolic chick thymus glucocorticoid receptor. Owing to the relatively fast dissociation of the glucocorticoid receptor-cortexolone complex, glucocorticoid receptor could be eluted with cortexolone as well as with triamcinolone acetonide from the affinity gel with similarly good yields. We obtained 75-150-fold purification factors (yield: 20-30%) using this column procedure. The partially purified glucocorticoid receptor was obtained in non-activated form. It had a Stokes radius of 6.2 +/- 0.1 nm. It could be activated to DNA-cellulose binding form by heat or 0.3 M KCl. KCl treatment activated 30-50% of the partially purified glucocorticoid receptor. Heat activation, however, was rather poor. Cortexolone-complexed, partially purified glucocorticoid receptor dissociated easily, and partially purified free glucocorticoid receptor, capable of steroid binding, could be obtained. Binding properties of the partially purified glucocorticoid receptor were then analyzed using different steroids. Dissociation rate constants were similar to those of the cytosolic glucocorticoid complexes. Association rate constants were consistently smaller than in the case of cytosolic glucocorticoid receptor, but the relative order of rates for different steroids was basically the same for glucocorticoid receptor in the two studied systems.

Effect of heat treatment on the glucocorticoid-receptor complex. Dependence on steroid structure.

The Arrhenius plot of heat inactivation of the rat liver glucocorticoid receptor protein gave a straight line with delta H++ = 115 kJ/mol over the 0-44 degrees C range. Molybdate ions were considerably protective but did not affect the linearity or slope of the Arrhenius plot. The effect of triamcinolone acetonide on heat stability of the receptor was similar to that of molybdate. On the other hand, glucocorticoid antagonists, although bound to the receptor, did not protect it from heat inactivation. Incubation of the complex of the glucocorticoid receptor with optimal glucocorticoids under activating conditions (elevated temperature or ionic strength) resulted in a considerable decrease in the dissociation rate. However, if the complex was incubated at 25 degrees C in the presence of molybdate, its dissociation rate did not change. Heat treatment without molybdate of complexes of glucocorticoid antagonists did not decrease the dissociation rate. These findings indicate that the decrease in dissociation rate is probably related to nucleophilic transformation. An 11 beta-hydroxyl group in the steroid structure seems to be an absolute requirement both for protection of the receptor against heat inactivation and for stabilization of the complex under conditions that promote nucleophilic transformation.

A new potent calmodulin antagonist with arylalkylamine structure: crystallographic, spectroscopic and functional studies.

An arylalkylamine-type calmodulin antagonist, N-(3, 3-diphenylpropyl)-N'-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine (AAA) is presented and its complexes with calmodulin are characterized in solution and in the crystal. Near-UV circular dichroism spectra show that AAA binds to calmodulin with 2:1 stoichiometry in a Ca(2+)-dependent manner. The crystal structure with 2:1 stoichiometry is determined to 2.64 A resolution. The binding of AAA causes domain closure of calmodulin similar to that obtained with trifluoperazine. Solution and crystal data indicate that each of the two AAA molecules anchors in the hydrophobic pockets of calmodulin, overlapping with two trifluoperazine sites, i.e. at a hydrophobic pocket and an interdomain site. The two AAA molecules also interact with each other by hydrophobic forces. A competition enzymatic assay has revealed that AAA inhibits calmodulin-activated phosphodiesterase activity at two orders of magnitude lower concentration than trifluoperazine. The apparent dissociation constant of AAA to calmodulin is 18 nM, which is commensurable with that of target peptides. On the basis of the crystal structure, we propose that the high-affinity binding is mainly due to a favorable entropy term, as the AAA molecule makes multiple contacts in its complex with calmodulin.

A novel specific binding site for anxiolytic homophthalazines in the rat brain.

Radioligand binding studies were performed in order to elucidate the mechanism of action of anxiolytic-neuroleptic homophthalazines. Rat striatal membrane preparations were found to bind 3H-EGIS 6775 [3H-GYKI-52 322, 3H-(1-(4-aminophenyl)-4-methyl-7,8-dimethoxy-5H-homophthalazine)] in a specific and displaceable manner. Several other brain regions tested were devoid of similar binding activity. Saturation analysis revealed that binding affinity was in the 10(-8)-10(-7) M range. Binding was enhanced by Mg2+ ions and, to a smaller extent by Ca2+ ions. The binding principle was sensitive to heat or trypsin treatment. This specific binding site appears, according to competition studies, different from the receptors whose presence in the rat striatum has been reported earlier.

Effect of calcium ion on the interaction between thrombin and heparin. Thermal denaturation.

Heat inactivation of thrombin at 54 degrees C followed first order kinetics with a rate constant of 1.0 min-1 approximately. Addition of heparin resulted in protection against thermal denaturation and, at the same time, rendered denaturation kinetics more complex. Analysis of the biphasic curve of heat inactivation in the presence of heparin revealed that the rate constants of the second phase changed systematically with heparin concentrations. Namely, at 4.5 x 10(-6)M, 9 x 10(-6)M, 1.8 x 10(-5)M and 3.6 x 10(-5)M heparin concentrations, the rate constants were 0.27 min -1, 0.17 min-1, 0.11 min-1 and 0.06 min-1, respectively. Sulfate as well as phosphate ions displayed also enzyme protection against heat inactivation, however, the same effect was obtained already at a heparin concentration, lower by three orders of magnitude. The kinetics of enzyme denaturation was not affected by calcium ions, whereas in the presence of heparin the inactivation rate of thrombin changed, i.e. calcium ions abolished the biphasic character of time course of thermal denaturation. Thus, the data suggest that calcium ions contribute to the effect of heparin on thrombin.

Autoradiographic localization and quantitative determination of specific binding sites of anxiolytic homophthalazines (formerly called 2,3-benzodiazepines) in the striato-pallido-nigral system of rats.

Homophthalazines (2,3-benzodiazepin-derivates, such as tofisopam, nerisopam, girisopam) constitute a drug family with strong anxiolytic and antipsychotic potencies. By autoradiography, all of these drugs showed a specific distribution pattern of binding sites exclusively in brain areas which relate to the striato-pallido-nigral system, while no specific label was found in any other brain areas in the rat. Quantitative analyses of the autoradiograms by computerized densitometry, as well as by a receptor binding assay on 32 microdissected brain areas showed very high concentrations of tritiated homophthalazines in the glubus pallidus, caudate nucleus, putamen and the substantia nigra. Relatively high density of binding sites was measured in the nucleus accumbens, the olfactory tubercle, the entopeduncular nucleus and the subthalamic nucleus. Concentrations measured in the cerebral cortical areas, cerebellum or brainstem nuclei did not differ from the background. No significant differences were found between the homophthalazines investigated in terms of the distribution patterns or density of binding sites.

Different neurorescue profiles of selegiline and p-fluoro-selegiline in gerbils.

The neuroprotective/neuronal rescue effects of selegiline are not exactly understood, and show great variability in clinical trials. In this study, the dose-dependence of neuronal rescue potency of selegiline and its analogue para-fluoro-selegiline (PFS) was investigated in gerbils. The compounds were tested in a transient global cerebral ischemia model. Selegiline expressed a bell-shaped, dose-response curve with high intrinsic activity (with greatest effect at 0.001 mg/kg), as opposed to PFS which shows a saturation profile. These findings indicate possible therapeutic differences between PFS and selegiline in the treatment of neurodegenerative disorders. Inhibition of progression of the disease (neuroprotective effect) and improvements of symptoms (MAO-B inhibition) may occur at the same dose level using PFS, while these doses are separated in case of selegiline.

A novel specific binding site for homophthalazines in the rat brain.

The specific binding sites of a homophthalazine, girisopam, in rat brain have been localized by qualitative and quantitative autoradiography. This substance exerts strong anxiolytic and antipsychotic effects both in rodents and in humans. High labeling was present in all major components of the extrapyramidal system, such as the caudate-putamen, globus pallidus, subthalamic nucleus, substantia nigra, and the extrapyramidal portion of the accumbens nucleus and the olfactory tubercle, while specific labeling was not seen in any other brain areas including the cerebral cortex, thalamus, cerebellum or brainstem areas. This novel distribution of girisopam is consistent with its antipsychotic effect and anxiolytic properties and may provide a morphological basis for further studies to elucidate the mechanisms of action of homophthalazines in the central nervous system.

3-Chloro-1,3,5-pregnatriene derivatives with glucocorticoid activity.

Novel synthetic glucocorticoid analogues were tested for receptor binding and glucocorticoid activity. They were of unusual structure, insofar as they had a 3-chloro rather than a 3-oxo function. 3-Chloro analogues of fluorinated glucocorticoids formed extremely stable complexes with the rat liver glucocorticoid receptor. 3-Chloro derivative of fluocinolone acetonide also had in vivo glucocorticoid activity. It induced tyrosine aminotransferase in the liver and repressed thymidine kinase in the thymus very effectively. It is concluded that 3-chloro analogues may retain glucocorticoid activity as well as the ability to bind to the glucocorticoid receptor protein.

[Molecular modeling studies of prolyl endopeptidase inhibitors]. / Prolil endopeptidáz inhibitorok molekulaszerkezeti vizsgálata.

Prolyl endopeptidase, a serine protease is considered to play an important role in the degradation of neuropeptides capable of changing the performance in learning and memory tasks in both animal and human. The inhibitors seem to be promising drug candidates to treat and prevent diseases with associated memory loss such as senile dementia. In the last decade advanced and improved new technologies have appeared to stimulate ideas in the design and synthesis of new drug molecules. The goal of this short communication is to review our results and observations, exemplified by our research on the inhibitors of prolyl endopeptidase. Among them qualitative and quantitative structure-activity relationship studies using conformational analyses, NMR measurements, pharmacophoric plots and CoMFA models are summarised.

Activation and deactivation of the glucocorticoid hormone-receptor complex.

Cytosol glucocorticoid receptors acquired the ability to bind to DNA-cellulose on incubation at 25-37 degrees C and/or in media of high ionic strength (0.3 M KCl). However, this activation was transient only. It was followed by deactivation whose rate was also dependent on incubation temperature and on the presence of potassium chloride. Deactivation resulted in a decreased but non-zero binding to DNA-cellulose. Specific triamcinolone acetonide binding to the receptor protein was not lost under the same conditions. Deactivation commenced before it became apparent, probably together with activation. Preactivated complex underwent deactivation even in conditions that would not allow significant decrease in DNA-cellulose binding without previous incubation at 37 degrees C. In contrast to previous reports it was found that fast activation/deactivation took place in the presence of 4 mM Ca2+. Molybdate ions slow down both activation and deactivation, but do not prevent activation by heat. Heat activated/deactivated complex differed in size from both non-activated and Ca2+-deactivated complexes. This finding suggests that heat and Ca2+-induced deactivation follow different mechanisms.

Effect of cross-linking on glucocorticoid receptor activation.

Chick thymus cytosol glucocorticoid receptor complexed with [3H]-triamcinolone acetonide was heat-activated after treatment with diimidates of varying chain length. Diimidates with maximal effective reagent length shorter than 0.5 nm influenced DNA-cellulose binding only slightly, whereas sebacic diimidate (maximal effective reagent length 1.21 nm) blocked activation completely. Coupled activation - deactivation process induced in the presence of calcium was again inhibited only by the longer diimidates. Sebacic diimidate treated complex had a molecular mass similar to that of the molybdate stabilized nonactivated complex, as judged by gel permeation chromatography. It is suggested that prevention of size reduction by cross-linking blocks receptor activation as well.

Kinetic deuterium isotope effects in glucocorticoid receptor activation.

Activation and deactivation of the chick thymus glucocorticoid receptor protein was studied in ordinary and heavy water by DNA-cellulose binding of the tritiated triamcinolone acetonide-receptor complex. Activation was significantly slower in heavy water if it was promoted by incubation at elevated temperature in buffers of low ionic strength. In the presence of 300 mM KC1 or after separation from the low molecular weight cytosol constituents, the complex was activated at the same rate in both solvents. Deactivation (time dependent loss of DNA-binding capacity) was much faster in ordinary than in heavy water regardless of gel filtration or the presence of KC1. A model of receptor activation-deactivation was constructed on the basis of these data that accounts for the observed kinetic deuterium isotope effects and reveals some submolecular details of the process.