On the Basicity of 8-Phenylsulfanyl Quipazine Derivatives: New Potential Serotonergic Agents.

A protonation state of serotonergic ligands plays a crucial role in their pharmacological activity. In this research, the basicity of 8-phenylsulfanyl quipazine derivatives as new potential serotonergic agents was studied. The most favorable protonation sites were determined in the gas and aqueous phases. In water, a solvation effect promoting the protonation of the N3 atom overcomes a positive charge delocalization phenomenon favoring a N1 atom protonation. The most stable conformations of neutral and protonated molecules in gas and water were found. It was demonstrated that a diprotonation reaction may occur. The most favorable among the diprotonated structures is the molecule with the N1 and N3 atoms protonated. A calculation of the pKa and pKa2 in water of a set of monosubstituted 8-phenylsulfanyl quipazine derivatives was performed using B3LYP/6-31G(d) and the SMD continuum solvation model. Enthalpic and entropic contributions to the pKa and pKa2 in gas and water were separated for a rationalization of a substituent effect on values of the pKa and pKa2. The relationship of the proton affinity and the solvation enthalpy in water with some reactivity descriptors, such as the Fukui function, the molecular electrostatic potential (MEP), and the global softness, was investigated. The order of the pKa values is the most controlled by the entropy. The diprotonation reaction, despite having an unfavorable enthalpy in water, is driven entropically. Final state effects in the diprotonated species were analyzed with the triadic formula. Results of a calculation of the theoretical basicity of the 8-phenylsulfanyl quipazines indicate that they should be monoprotonated on the N3 atom in the CNS environment. Diprotonation of the studied compounds may occur in very acidic body fluids such as the gastric juice.

Experimental and theoretical studies on the molecular properties of ciprofloxacin, norfloxacin, pefloxacin, sparfloxacin, and gatifloxacin in determining bioavailability.

The aim of this investigation is to identify, by in silico and in vitro methods, the molecular determinants, e.g., solubility in an aqueous medium and lipophilic properties, which have an effect on the bioavailability of five selected fluoroquinolones. These properties were estimated by analysis of the electrostatic potential pattern and values of free energy of solvation as well as the partition coefficients of the studied compounds. The study is based on theoretical quantum-chemical methods and a simple experimental shake-flask technique with two immiscible phases, n-octanol and phosphate buffer. The solvation free energy values of compounds in both environments appeared to be negative. The wide range of electrostatic potential from negative to positive demonstrates the presence of dipole-dipole intermolecular interactions, while the high electron density at various sites indicates the possibility of hydrogen bond formation with solvent molecules. High partition coefficient values, obtained by summing the atomic contributions, did not take various correction factors into account and therefore were not accurate. Theoretical partition coefficient values based on more accurate algorithms, which included these correction factors (fragmental methods), yielded more accurate values. Theoretical methods are useful tools for predicting the bioavailability of fluoroquinolones.

Novel renin inhibitors containing a non-peptide aminoalkanoyl moiety at P1-P1' position.

Six novel potential renin inhibitors have been designed and synthesized. All these inhibitors contained an unnatural aminoalkanoyl moiety at the central position P1- P1' of the molecule, which is attacked by renin. The moiety consists of pseudodipeptidic units, transition state analogues of a natural dipeptide of the parent substance: 4-amino-3-hydroxybutanoic acid (AHBA), 4-amino-5-(4-ethoxyphenyl)-3-hydroxypentanoic acid (AEPHPA), 4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA) or 4-amino-3-hydroxynonanoic acid (AHNA). An unnatural moiety, 4-methoxyphenylalanylhistydyl (Phe(4-OMe)-His) has been introduced at the P3-P2 position of the obtained compounds. Five compounds contain isoamylamide of 6-aminohexanoic acid (epsilon-Ahx-laa) at the P2'-P3' position. One of designed inhibitors has been obtained in the form of an ethyl ester. The in vitro renin inhibitory activity of all synthesized compounds is contained within the range 10(-6) - 10(-8) M. The compound in the form of an ethyl ester has proven to be the most active (IC50 = 1.3 x 10(-8) M) but also susceptible to enzymatic degradation. The other five inhibitors were stable to chymotrypsin.

A nicked duplex decamer DNA with a PEG(6) tether.

A dumbbell double-stranded DNA decamer tethered with a hexaethylene glycol linker moiety (DDSDPEG), with a nick in the centre of one strand, has been synthesised. The standard NMR methods, E.COSY, TOCSY, NOESY and HMQC, were used to measure (1)H, (31)P and T:(1) spectral parameters. Molecular modelling using rMD-simulated annealing was used to compute the structure. Scalar couplings and dipolar contacts show that the molecule adopts a right-handed B-DNA helix in 38 mM phosphate buffer at pH 7. Its high melting temperature confirms the good base stacking and stability of the duplex. This is partly attributed to the presence of the PEG(6) linker at both ends of the duplex that restricts the dynamics of the stem pentamers and thus stabilises the oligonucleotide. The inspection of the global parameters shows that the linker does not distort the B-DNA geometry. The computed structure suggests that the presence of the nick is not disturbing the overall tertiary structure, base pair geometry or duplex base pairing to a substantial extent. The nick has, however, a noticeable impact on the local geometry at the nick site, indicated clearly by NMR analysis and reflected in the conformational parameters of the computed structure. The (1)H spectra also show much sharper resonances in the presence of K(+) indicating that conformational heterogeneity of DDSDPEG is reduced in the presence of potassium as compared to sodium or caesium ions. At the same time the (1)H resonances have longer T:(1) times. This parameter is suggested as a sensitive gauge of stabilisation.

Clonidine hypotension in spontaneously hypertensive rats (SHR) depends on the functional state of GABAergic and glutamatergic systems.

The effect of gamma-aminobutyric acid (GABA)A receptors and of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor blockade on clonidine hypotension was studied. The experiments were performed on spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. We found that the blockade of GABAA receptors line significantly (P < 0.01) reduced hypotensive responses to clonidine. Similarly, the NMDA receptor antagonist dizocilpine (MK-801) completely abolished the blood pressure lowering effect of clonidine. Our findings support the conclusion that clonidine hypotension is closely related to the functional state of both inhibitory GABAergic and excitatory glutamatergic systems.

The efficacy and potency of antiplatelet activity of ticlopidine is increased by aspirin.

Concomitant therapy with ticlopidine (T) and aspirin (ASA) profoundly increases spectrum of antiplatelet activities of both drugs. It was hypothesized that in addition to increased spectrum of activity (efficacy) each drug may potentiate the specific antiplatelet activity (potency) of the other. In 32 volunteers whole blood platelet aggregation (PA) in response to ADP, collagen, and arachidonic acid was evaluated ex vivo following 10-day treatments with normal or subthreshold doses of T or ASA with addition of second drug on the 5th day of administration of the first. PA was measured before, on day 5 and 10 of treatment. The results indicate that ASA increased spectrum of activity of T, i.e. T and ASA in combination, were significantly more effective against collagen-induced PA than either drug alone. This increased efficacy was retained when subthreshold dose of T (100 mg, qd) was used. T was without effect on AA-induced and ASA on ADP-induced PA. However, ASA potentiated effect of T on ADP-induced PA; the subthreshold dose of T (100 mg, qd) in presence of ASA (100 mg, qd) exerted powerful inhibition. Thus, combination therapy increases both efficacy and potency of T allowing for reduction of the dose.

Pharmacodynamics of ticlopidine: relation between dose and time of administration to platelet inhibition.

In spite of long clinical experience with ticlopidine (T) knowledge of its pharmacodynamics is limited. In this study relation between dose and time of administration of T to platelet inhibition was investigated in 62 healthy volunteers ex vivo in whole blood and platelet rich plasma. Gender-related sensitivity of platelets to ticlopidine was also evaluated. Inhibition of ADP-induced platelet aggregation by T, 500 mg, daily, was almost identical in both sexes. 100 mg daily did not inhibit ADP-induced platelet aggregation even after 14 days of administration. 250 mg daily induced strong inhibition on day 5 of administration comparable to the inhibition obtained with 500 mg daily dose. The antiplatelet (ADP) effect of T (500 mg, daily) was present on day 2-3 and full inhibitory effect on day 4 of administration. T1/2 of antiplatelet (ADP) activity of T was 5.3 days and full recovery of platelets activity 11-13 days. No rebound phenomenon was present. T (regardless the dose) inhibited platelet aggregation induced by small but not high concentrations of collagen and was without effect on arachidonic acid-induced platelet aggregation. Therefore, T is not suitable for treatment of acute event, 250 mg daily dose should be used especially for combination with other drugs and 11 days washout interval seems necessary to change the treatment or to perform surgery.

Evaluation of radioiodinated histamine as isotope carrier in vivo.

The iodo derivative of histamine labelled with 125I has been used for many years to prepare tracers used in RIA systems. The aim of this study was to evaluate radioiodinated histamine as a potential isotope carrier for in vivo applications. The biological behaviour of radioiodinated histamine has been investigated in rodents. The observed absence of any specific iodohistamine uptake by a critical organ or tissue promises a very quick distribution of the iodohistamine in soft tissues, and a rapid rate of whole-body clearance via the urinary tract (e.g. over 50% of the injected dose (ID) during the first hour after administration). In spite of moderately low in vitro stability of iodohistamine in serum, biodistribution studies in rodents have not shown any significant release of iodine from the parent molecule in the whole animal. Low uptake was observed in the thyroid (e.g. 0.22 and 0.11% ID at 1 and 2 h after administration to rats), and not more than 3% of injected activity was detected in the stomach in all of the biodistribution experiments. Moreover, our results refute any possibility of competition between histamine and iodohistamine for receptor binding sites, and suggest that radioactive mono-iodohistamine may be used successfully to develop some new radiolabelled bioactive molecules with potential application in vivo.

Mitochondrial activity after cold preservation of pancreatic islet cells treated with pefloxacin (PFX).

Mitochondrial energetic and oxidative dysfunctions caused by free radical production trigger release of proinflammatory cytokines involved in organ rejection. The aim of this study was to investigate the role of a fluoroquinolone drug, pefloxacin (PFX) and those of various cold preservation solutions on pancreatic beta cell viability. Our data clearly demonstrate that islet cell viability, as determined by glucose-stimulated insulin secretion, is directly correlated with reduced expression of microsomal cytochrome P-450IIIA. Moreover, IL-2, a known mediator of apoptosis was found to be downregulated, whereas TNF-alpha had been upregulated for the first 18 hours after pefloxacin administration. These results demonstrate that pefloxacin downregulates the expression of cytochrome P-450IIIA isozyme and regulates the production of TNF-alpha and IL-2. Thus, we postulate that the presence of pefloxacin in the pancreatic islet cells before organ preservation facilitates increased cell viability.

Monitoring of rat islet allografts with dithizone after induction of donor specific transplant tolerance by intrathymic administration of soluble alloantigens.

Transplantation of whole pancreas or pancreatic islets remains a promising approach to treatment of diabetes mellitus. Since there is no efficient method presently known for in vivo detection of pancreatic islet rejection, we have utilized dithizone [DTZ] to monitor the survival of transplanted islet allografts following the induction of tolerance by a new strategy of deliberate introduction of donor antigens into the adult thymus. In this study, we examined the morphology of islet allografts in vivo and in vitro following pretreatment with intrathymic (IT) inoculation of 2 mg soluble Ag obtained from 3M KCl extracts of resting T-cells with or without ALS immunosuppression in the WF-to-Lewis combination. Fresh isolated rat islets stained pink 3-5 minutes following exposure to medium containing 0.12 mM DTZ solution in DMSO. Intravenous (i.v.) injection of DTZ solution into unmodified recipients of islet allografts that had rejected their grafts showed massive degranulation of islets which did not stain pink with DTZ. This was confirmed by microscopic finding of fibrosis and lymphocytic infiltration. In contrast, i.v. injection of DTZ solution into long-term recipients of islet allografts at 50, 100, and 150 days after transplantation showed viable islet cells which stained crimson red with DTZ and the findings were confirmed with microscopic sections. This study demonstrates that DTZ is an effective means of in vivo and in vitro identification of transplanted pancreatic islets and suggests that this strategy may have potential clinical application in the diagnosis of the pancreatic islet rejection.

Theoretical studies on molecular determinants for recognition at H3 histamine receptors.

The determinants for recognition at H3 histamine receptors are considered. Findings based on quantum-chemical calculations suggest that H3 histamine receptor is less hydrophilic than the H2. The form most likely to be recognized by the H3 receptor is an intramolecularly hydrogen-bonded form of alpha-methylhistamine. Receptor environment and hydration effects of active form of histamine analogs are of crucial importance.

Studies on activation of H3 histamine receptor: a mechanistic model.

The recognition and the activation mechanism of the H3 histamine receptor was studied based on quantum-chemical calculations. A mechanistic model proposed both for recognition and activation stage clarifies different properties of histamine and alpha-methylhistamine at the H3 receptor. Interaction with a hypothetical receptor sites leads to the opening of the intramolecular hydrogen bonding in histamine, whereas the alpha-methylhistamine remains in closed conformation.

The synthesis, radioiodination and preliminary biological study of the new carboxylic derivatives of dithizone.

Synthesis, characteristics and radioiodination of the new carboxylic derivatives of dithizone are described in this paper. We have applied the carboxy dithizones for preparation of radioactive compounds by coupling with [131I]-histamine. Preliminary biological studies of the new radiodithizone were done in rats after two different application routs: peripheral i.v. injection and direct injection to splenic artery. Biodistribution of the carboxy dithizone-[131I]-histamine conjugate (i.v. injection) was quite different than that for free [131I]-histamine. However, uptake of activity in pancreas was low (0.81% g-1 of tissue). Direct application of the conjugate to splenic artery resulted in high activity retention in pancreas after 30 and 45 min post injection (respectively 8.8 and 12.4% g-1 of tissue) indicating potential usefulness of the new radiodithizone for in vivo monitoring of pancreas.

Identification of transplanted pancreatic islet cells by radioactive dithizone-[131I]-histamine conjugate. Preliminary report.

BACKGROUND: The unique mechanism of dithizone action in the interior of the viable pancreatic islet suggests the possible development of a specific radiopharmaceutical that may have a potential clinical application in the diagnosis of the pancreatic organ allografts or islets rejection. The radiodiagnostic properties of the newly developed radioactive analogue of dithizone, i.e. Dithizone-[(131)I]-Histamine conjugate have been evaluated in the present study. METHODS: The four islet cells transplantation models were chosen for this purpose. The most important feature of the Dithizone-[(131)I]-Histamine conjugate is its possessed ability of zinc chelation. As was presented in the recent study, the conjugate stains pink-reddish the isolated pancreatic islets in vitro. Among the studied transplantation models, only the islets grafting under testis capsule enabled determination of the pancreatic islets in rats by radioactive Dithizone-[(131)I]-Histamine conjugate. The level of the radioactivity in the recipient testis (right) was almost two times higher compared to the controls (0.24 vs. 0.13% ID/g, respectively). CONCLUSIONS: These preliminary data demonstrate the ability of the developed radioactive analogue of dithizone for in vivo identification of transplanted pancreatic islets, and suggests a potential clinical application of the radiodithizone in the diagnosis of the pancreatic islet rejection.

Biological properties of genistein. A review of in vitro and in vivo data.

Genistein--a soy derived isoflavone has recently attracted much attention of the medical scientific community. This compound was found to be a potent agent in both prophylaxis and treatment of cancer as well as other chronic diseases. The great interest that has focused on genistein led to the identification of numerous intracellular targets of its action in the live cell. At the molecular level, genistein inhibits the activity of ATP utilizing enzymes such as: tyrosine-specific protein kinases, topoisomerase II and enzymes involved in phosphatidylinositol turnover. Moreover, genistein can act via an estrogen receptor-mediated mechanism. At the level one step higher, i.e., at the cellular level, genistein induces apoptosis and differentiation in cancer cells, inhibits cell proliferation, modulates cell cycling, exerts antioxidant effects, inhibits angiogenesis, and suppresses osteoclast and lymphocyte functions. These activities make genistein a promising innovative agent in the treatment of cancer. Additionally, genistein health beneficial effects have been shown in osteoporosis, cardiovascular diseases and menopause. Genistein was also successfully used as an immunosuppressive agent both in vitro and in vivo. All these effects at the three biological levels of action need varied genistein concentrations and only some of them are relevant in people consuming soy-rich diet. The others would occur after purified genistein administration at higher doses. The main genistein advantage as a potential drug is its multidirectional action in the live cell and its very low toxicity.

Anticancer activity of genistein-piperazine complex. In vitro study with HL-60 cells.

Genistein, a principal soy isoflavone, has recently aroused interest in medical research owning to its numerous biochemical properties such as: inhibition of the activity of tyrosine-specific protein kinases and topoisomerase II, estrogenic and antioxidant activity as well as antiproliferative and antiangiogenic effects. Therefore, genistein is extensively investigated as a novel anticancer drug. To improve physicochemical properties of genistein (e.g., water solubility) we have synthesized its complexes with amines. Genistein-piperazine complex (GP) has been then examined whether it exhibits anticancer action against human promyelocytic leukemia cell line (HL-60) cultured in vitro. The parallel study with pure genistein has also been undertaken. Cell proliferation, viability, apoptosis and cell cycle kinetics have been assayed for various drugs concentrations (10-40 microM) and periods of exposure (1-6 days). GP reduced proliferation rate, decreased cell viability and induced apoptotic cell death, in a dose- and time-dependent manner. Flow-cytometric analysis of cell cycle distribution revealed a progressive and sustained accumulation of cells in the G2/M phase that was accompanied by unperturbed protein synthesis. The measured anticancer effects of GP and genistein were qualitatively and quantitatively similar, indicating that genistein-amine complex does not loose the activity of the parent compound.

NMR studies on three optical active drug molecules.

NMR spectroscopy is one of very few methods that enable deep insight into molecular structure and distinguishing of enantiomers. It usually requires the use of optically active agents that differentiate S and R forms through an interaction with studied molecule differentiate S and R forms. The aim of the present studies was to perform a preliminary NMR study on three chiral drugs: Fluoxetine hydrochloride, Ibuprofen, and Zolmitriptan by use of three optical solvating agents: R(-) 2,2,2-trifluoro-1-(9-anthryl)ethanol (I), (R)(+) 1,1'-bi-2-naphthol (II), and (S) tert-butyl-phenylphosphinothioic acid (III).

Inhibition of primates performed xenoantibody binding to pig aortic endothelial cells and its potential application to xenotransplantation.

IgM xenoantibodies are believed to play the most important role in the hyperacute rejection of distantly related species. The purpose of this study was to determine whether DL-Penicillamine could inactivate binding and cytotoxicity of adult baboon performed xenoantibodies to pig endothelial cells. Different concentrations of DL-Penicillamine were used to treat pooled baboon serum over various lengths of time. In order to determine the reactivity of baboon natural xenoantibodies to pig endothelial cells complement-mediated cytotoxicity assay was used. ELISA assay was used to assess IgM and IgG binding to pig endothelial cells. Subsequently DL-Penicillamine was dialyzed to determine its potential clinical application. Results indicate that baboon performed xenoantibodies class IgM and IgG bind to pig endothelial cells, but only IgM xenoantibody is cytotoxic DL-Penicillamine treatment can significantly reduce cytotoxicity and eliminate binding of IgM xenoantibodies to pig endothelial cells despite continued binding of IgG xenoantibodies to pig endothelial cells. In addition, DL-Penicillamine can be dialyzed, suggesting that it may be applicable in xenotransplantation and its toxicity can be significantly reduced by routine hemodialysis.

Iodo-derivatives of dithizone with potential diagnostic application.

The mono and diiodo-derivatives of dithizone labelled with radioactive 131I isotope were obtained. Those compounds can be used for diagnostic of pancreas and other organs to monitor pathological states.

Lidocaine as a protective agent during pancreas cold ischemia.

Pancreatic islet transplantation in humans is a promising alternative for substitutive insulin therapy of IDDM (Insulin Dependent Diabetes Mellitus). Storage of harvested organs is a one of the most important factors, which influence efficacy of islet isolation process. In this sense, appropriate pancreas storage is the main point the successful pancreatic islet isolation. The purpose of the present study was to find out whether lidocaine, a well known membrane stabilizer and PLA2 (phospholipase A2) inhibitor could be applied in pancreas preservation for protection of endo- and exocrine pancreatic tissue from cells damage which occurs during and after storage. For this purpose, the effects of lidocaine on 1) viability and 2) endocrine function of pancreatic islets, isolated from pancreases exposed to cold ischemia, were investigated in this study. Our study showed hat lidocaine, injected intraductally before pancreas harvesting, improves efficacy of islet isolation. We found that the yields of islets in the groups treated with lidocaine were significantly higher when compared with controls. Glucose challenge test performed on these islets indicated that after the treatment with lidocaine, islets were more sensitive to glucose stimulation when compared with control islets, although the metabolic activity estimated by MTT test was comparable in both groups. In summary, donor pretreatment with lidocaine seems to be the safe method of protection of preserved pancreases from cell damage, caused by membranes destruction during cold ischemia.