Showing papers in "Methods and Findings in Experimental and Clinical Pharmacology in 2002"

Standardized low-resolution brain electromagnetic tomography (sLORETA): technical details.

Abstract: Scalp electric potentials (electroencephalograms) and extracranial magnetic fields (magnetoencephalograms) are due to the primary (impressed) current density distribution that arises from neuronal postsynaptic processes. A solution to the inverse problem--the computation of images of electric neuronal activity based on extracranial measurements--would provide important information on the time-course and localization of brain function. In general, there is no unique solution to this problem. In particular, an instantaneous, distributed, discrete, linear solution capable of exact localization of point sources is of great interest, since the principles of linearity and superposition would guarantee its trustworthiness as a functional imaging method, given that brain activity occurs in the form of a finite number of distributed hot spots. Despite all previous efforts, linear solutions, at best, produced images with systematic nonzero localization errors. A solution reported here yields images of standardized current density with zero localization error. The purpose of this paper is to present the technical details of the method, allowing researchers to test, check, reproduce and validate the new method.

Functional imaging with low-resolution brain electromagnetic tomography (LORETA): a review.

Abstract: This paper reviews several recent publications that have successfully used the functional brain imaging method known as LORETA. Emphasis is placed on the electrophysiological and neuroanatomical basis of the method, on the localization properties of the method, and on the validation of the method in real experimental human data. Papers that criticize LORETA are briefly discussed. LORETA publications in the 1994-1997 period based localization inference on images of raw electric neuronal activity. In 1998, a series of papers appeared that based localization inference on the statistical parametric mapping methodology applied to high-time resolution LORETA images. Starting in 1999, quantitative neuroanatomy was added to the methodology, based on the digitized Talairach atlas provided by the Brain Imaging Centre, Montreal Neurological Institute. The combination of these methodological developments has placed LORETA at a level that compares favorably to the more classical functional imaging methods, such as PET and fMRI.

Transforming growth factor-beta 1-induced collagen production in cultures of cardiac fibroblasts is the result of the appearance of myofibroblasts.

Abstract: Transforming growth factor-beta 1 (TGF-beta 1), which appears in high concentrations in fibrotic cardiac tissue, is a potent inductor of tissue collagen deposition and of the differentiation of fibroblasts to myofibroblasts. It is accepted that TGF-beta 1 is a potent stimulator of collagen secretion by fibroblasts. The aim of the present study was to determine which type of cells, fibroblasts and/or myofibroblasts are stimulated, in terms of collagen production, by TGF-beta 1. Therefore, using cultures of second-passage rat cardiac fibroblasts, we investigated the dose- (0.003-15 ng/ml) and time-dependence (2-48 h) of the TGF-beta 1-induced effects on collagen production and on the appearance of myofibroblasts, as estimated by the presence of alpha-smooth muscle actin (alpha-SMA; a marker of myofibroblasts). The reversibility of the TGF-beta 1-stimulated effects was also studied. The dose- and time-dependent stimulation of collagen production was closely associated with the induction of alpha-SMA. TGF-beta 1 did not change the cell phenotype or increase collagen production in rat cardiac fibroblasts cultures after a long incubation (24-28 h) at low concentrations (< 1 ng/ml), or after a short incubation (2-4 h) at high concentrations (1-15 ng/ml). However, after a long incubation at high concentrations, TGF-beta 1 changed the cell phenotype and increased collagen production in these cultures through the differentiation of fibroblasts to myofibroblasts. A maximal increase of collagen production (two-fold, p < 0.001) was observed after incubation of fibroblasts with 15 ng/ml TGF-beta 1 for 48 h. Under these conditions, alpha-SMA was increased by 3.5-fold (p < 0.001) and second-passage cultures of fibroblasts and their offspring in the next passage consisted mainly of myofibroblasts. The stimulation of collagen by 15 ng/ml TGF-beta 1 for 48 h was irreversible. In fact, additional incubation of these second-passage TGF-beta 1-stimulated cultures without TGF-beta 1 for 2 days did not decrease the high activity of collagen production. Moreover, the third-passage offspring of these TGF-beta 1-stimulated fibroblasts cultured without TGF-beta 1 also showed a higher production of collagen compared with control fibroblasts. Furthermore, the increased collagen production in the third-passage fibroblast offspring of the second-passage TGF-beta 1-stimulated fibroblasts could not be further stimulated by TGF-beta 1. Thus, the activity of collagen production in TGF-beta 1-stimulated cultures and in their next passage offspring is not sensitive to TGF-beta 1. Our data suggest that TGF-beta 1-stimulated collagen production in cultures of adult rat cardiac ventricular fibroblasts cannot be explained by a direct stimulation of collagen production, either in fibroblasts or in myofibroblasts. Instead, TGF-beta 1 induces differentiation of fibroblasts to myofibroblasts, the latter having a higher activity for collagen production than the former.

Classification and evaluation of the pharmacodynamics of psychotropic drugs by single-lead pharmaco-EEG, EEG mapping and tomography (LORETA).

Abstract: Utilizing computer-assisted quantitative analyses of human scalp-recorded electroencephalogram (EEG) in combination with certain statistical procedures (quantitative pharmaco-EEG) and mapping techniques (pharmaco-EEG mapping), it is possible to classify psychotropic substances and objectively evaluate their bioavailability at the target organ: the human brain. Specifically, one may determine at an early stage of drug development whether a drug is effective on the central nervous system (CNS) compared with placebo, what its clinical efficacy will be like, at which dosage it acts, when it acts and the equipotent dosages of different galenic formulations. Pharmaco-EEG profiles and maps of neuroleptics, antidepressants, tranquilizers, hypnotics, psychostimulants and nootropics/cognition-enhancing drugs will be described in this paper. Methodological problems, as well as the relationships between acute and chronic drug effects, alterations in normal subjects and patients, CNS effects, therapeutic efficacy and pharmacokinetic and pharmacodynamic data will be discussed. In recent times, imaging of drug effects on the regional brain electrical activity of healthy subjects by means of EEG tomography such as low-resolution electromagnetic tomography (LORETA) has been used for identifying brain areas predominantly involved in psychopharmacological action. This will be demonstrated for the representative drugs of the four main psychopharmacological classes, such as 3 mg haloperidol for neuroleptics, 20 mg citalopram for antidepressants, 2 mg lorazepam for tranquilizers and 20 mg methylphenidate for psychostimulants. LORETA demonstrates that these psychopharmacological classes affect brain structures differently.

Studies on the antiplasmodial properties of some South African medicinal plants used as antimalarial remedies in Zulu folk medicine.

Abstract: The parasite lactate dehydrogenase (pLDH) assay method, a recently developed in vitro enzymatic method for evaluating antimalarial compounds, was used to examine the antiplasmodial activities of the aqueous leaf, stem-bark and fruit extracts of some plants used for the treatment and/or prophylaxis of malaria in KwaZulu-Natal province of South Africa. The in vitro antiplasmodial assay was carried out using a chloroquine-sensitive strain of malarial parasite, Plasmodium falciparum D10. A preliminary phytochemical analysis of the plant extracts was carried out using UV spectral analysis and thin-layer chromatography (TLC) to separate the chemical constituents of the extracts. Their chemical components were subsequently identified by treating the TLC plates with various spray reagents. Of the 14 plant extracts investigated, only 10 were found to have IC50 values of 10-50 micrograms/ml. The two most active extracts were Psidium guajava stem-bark extract and Vangueria infausta leaf extract, both of which showed IC50 values of 10-20 micrograms/ml. Phytochemical analysis of these two active plant extracts revealed the presence of anthraquinones, flavonoids, seccoirridoids and terpenoids.

Pharmacological studies on Myristica fragrans--antidiarrheal, hypnotic, analgesic and hemodynamic (blood pressure) parameters.

Abstract: Recurrent diarrhea is prevalent in developing countries, particularly in tropical regions. A natural based antidiarrheal home remedy can serve as an ideal health tool to limit diarrhea-related morbidity and mortality. In the traditional Indian medical science of Ayurveda, nutmeg is one such plant said to possess antidiarrheal activity. A study was therefore planned to assess the various pharmacological effects (antidiarrheal, sedative, analgesic and blood pressure) of nutmeg. Both Nutmeg crude suspension (NMC) and petroleum ether (PE), but not aqueous extract (Aq), decreased the mean number of loose stools or increased the latency period. NMC increased intestinal tone while PE had no such effect. PE had no effect on guinea pig ileum, but inhibited the contraction produced by acetylcholine, histamine and prostaglandin. NMC but not PE extract showed a significant but weak analgesic effect. While PE effectively potentiated both phenobarbitone and pentobarbitone-induced sleeping time, NMC was considerably less effective. NMC administered intraduodenally did not produce much effect on blood pressure (BP), but potentiated the action of exogenously administered adrenaline and nor-adrenaline. On the other hand, PE in higher, but not lower, doses caused a precipitous fall in BP not blocked by atropine. Thus, overall extracts of nutmeg showed a good antidiarrheal effect, with a significant sedative property. The extracts possessed only a weak analgesic effect, with no harmful effects on blood pressure and ECG.

Pica in mice as a new model for the study of emesis.

Abstract: In general, rats and mice have not been used in research on emesis because they do not vomit. However, emetogenic stimuli such as anticancer drugs, apomorphine, copper sulfate and rotation induced pica, a behavior characterized by eating nonfood substances such as kaolin, in rats. We also found that cisplatin induced pica in mice, but it was rather difficult to determine the exact kaolin consumption in this species. In this study, we prepared kaolin pellets mixed with carmine, a dye not absorbed in the gastrointestinal tract, and estimated kaolin consumption by determination of carmine excreted in feces. Cisplatin (5 mg/kg) caused a significant increase in kaolin consumption (saline: 0.15 +/- 0.08 g vs. cisplatin: 0.45 +/- 0.16 g) and pretreatment with the 5-HT3 receptor antagonist, ondansetron (2 mg/kg), suppressed the increased consumption (vehicle: 0.33 +/- 0.05 g vs. ondansetron: 0.13 +/- 0.04 g). These findings suggested that the exact kaolin consumption could be quantified by the determination of carmine in feces and that mice could be useful for studying emesis.

Rat models of genetic absence epilepsy: what do EEG spike-wave discharges tell us about drug effects?

Abstract: Electroencephalographic studies in the WAG/Rij rats of Nijmegen and genetic absence epileptic rats of Strasbourg (GAERS), two genetic models for human generalized absence epilepsy, illustrate the usefulness of drug-electroencephalogram (EEG) interaction studies. In the EEG of both types of rats, spontaneously occurring spike-wave discharges are present. For drug discovery, a model with predictive validity is imperative, and both the WAG/Rij and the GAERS models seem adequate. The present paper discusses effects on spike-wave discharges of various compounds that are clinically used. Not only new antiepileptic drugs, such as remacemide, loreclezole, lamotrigine, tiagabine, gabapentin, progabide and levetiracetam are evaluated, but also drugs used for other purposes, such as etomidate and fentanyl-fluanisone for anesthesia, opioidergic drugs and drugs used for strokes. It is shown that some new antiepileptic drugs, such as tiagabine, have spike-wave discharge-increasing properties, while other drugs are worth studying in clinical trials for antiabsence treatment. Furthermore, it is shown that many commonly used drugs such as analgesics, anesthetics and drugs to treat stroke generally enhance spike-wave discharges. It can be concluded that EEG monitoring is imperative for the discovery and development of potentially antiepileptic compounds and thin genetic rat models such as the WAG/Rij or GAERS, to a large extent, can reliably predict clinical efficacy of various types of compounds as well as alert us of potentially adverse effects.

Influence of lipophilicity and lysosomal accumulation on tissue distribution kinetics of basic drugs: a physiologically based pharmacokinetic model.

Abstract: This paper examines the role of lipophilicity in the tissue distribution kinetics of basic drugs. Basic drugs have a large distribution volume and are distributed widely in various tissues in the following order: lung, fat, heart, kidney, brain, gut, muscle and bone. The fat volume in the whole body influences the disposition kinetics. There is a good correlation in various tissues between the tissue-plasma concentration ratio and the octanol-water partition coefficient among various drugs. We constructed a physiologically-based pharmacokinetic model on the basis of drug lipophilicity and found that drug distribution decreased when NH4Cl was administered concomitantly. In regards to subcellular distribution, the relative specific contents of chlorpromazine, imipramine and biperiden with respect to the protein in lysosomes were 7.3, 9.6 and 4.2, respectively, while those in other subcellular organella, including mitochondria, were only 0.4-1.7, indicating preferential accumulation of these drugs in lysosomes. The uptake of basic drugs into lysosomes depended on both intralysosomal pH and drug lipophilicity. As the lipophilicity of the basic drugs increased, they accumulated more than would have been predicted from the pH-partition theory and raised the intralysosomal pH more potently, probably owing to their binding with lysosomal membranes, with or without intralysosomal aggregation. We conclude that the distribution kinetics of basic drugs is driven by drug lipophilicity and uptake into lysosomes, and these phenomena provide a possible basis for drug interaction in clinical treatments.

Studies on gastrointestinal tract functional changes in diabetic animals.

Abstract: Diarrhea and constipation are frequently reported gastrointestinal complications in diabetic patients. These disorders seem to be the consequence of altered innervations of a receptor system in the gastrointestinal tract in diabetes. We investigated the functional changes of cholinergic and beta-adrenergic receptor activities in rat ileum tissue after 8 weeks of control and streptozotocin (STZ)-induced diabetes in rats. Functional changes with different agonists were observed in diabetes: In diabetic rat ileum, carbachol increased the maximal contraction without changing pD2 values, whereas with acetylcholine, no changes were observed in the maximal contractile (Emax) response and pD2 values in diabetes were comparable with controls. With the beta-adrenoceptor agonists isoproterenol and salmeterol, decreased maximal relaxation responses were observed in 8-week diabetic rat ileums as compared with age-matched controls. With isoproterenol, pD2 values were also decreased in diabetic ileum, although not with salmeterol. On the other hand, with the in vivo charcoal meal test, the percentage of transit was increased in diabetic mice. The antitransit percentage was significantly increased in diabetic mice with the muscarinic antagonist atropine and the beta 2-selective agonist salbutamol, as compared with age-matched control mice. Results obtained from these in vivo and in vitro studies indicate that gastrointestinal complications such as diabetic diarrhea may occur because of functional changes such as increased intestinal transit and decreased intestinal tone due to increased cholinergic and decreased beta-adrenergic receptor activities in diabetes mellitus.

Structural and energetic processes related to P300: LORETA findings in depression and effects of antidepressant drugs.

Abstract: Noninvasive electrophysiological neuroimaging applied to cognitive components of event-related potentials (ERPs) may differentiate between structural and energetic processes related to information processing. The structural level, revealed by the location of the local maxima of the current source density distribution, describes the time-dependent network of activated brain areas. The magnitude of the source strength, a measure of the energetic component, describes the allocation of processing resources. ERPs were recorded in an odd-ball paradigm and low-resolution brain electromagnetic tomography (LORETA) was applied for standard and target ERP components. In a group of 60 menopausal depressed patients of 45-60 years of age, reduced P300 source strength was observed bilaterally, temporally and medially prefrontally reaching to rostal parts of the anterior cingulate, compared with 29 age-matched controls. In a double-blind, placebo-controlled study, 2 mg of the antidepressant citalopram induced a significant increase of P300 source strength in the (left) prefrontal cortex and precuneus compared with placebo, reaching to the posterior cingulate. Similar increases were observed after 800 mg S-adenosyl-L-methionine (SAMe) administered intravenously in ten young healthy subjects aged 22-33, and they were even more pronounced in ten elderly healthy subjects aged 56-71. Thus, ERP-tomography identified changes in energetic sources in brain areas predominantly involved in depression and in antidepressant action.

Pyrogallol-induced hepatotoxicity in rats: a model to evaluate antioxidant hepatoprotective agents.

Abstract: Various hepatic disorders and hepatotoxic agents are associated with increased free radical generation. In the present study, the free radical generator pyrogallol (100 mg/kg i.p.) caused significant hepatic damage. The serum enzymes asparatate aminotransaminase (AST) and alanine aminotransaminase (ALT) increased to 357 +/- 30.7 IU/I and 147.8 +/- 28.4 IU/I, respectively in the pyrogallol-treated group compared with 208.4 +/- 4.1 IU/I and 84.5 +/- 19.5 IU/I, respectively in the control rats. Compared with control rats, the liver tissue in the pyrogallol-treated group showed an increased level of malondialdehyde (MDA) as well as glutathione (GSH). The infiltration of white blood cells into the liver tissue, as seen histologically, further substantiated liver damage. Pretreatment with a standard hepatoprotective drug (silymarin, 100 mg/kg i.p.) afforded significant protection against pyrogallol hepatotoxicity, as evidenced by amelioration of the raised serum markers of hepatic function, markers of oxidative stress and normal liver histology. Thus, pyrogallol-induced hepatotoxicity could be used as an appropriate model to evaluate hepatoprotective agents that have an antioxidant property.

Identifying responders to serotonin agonists by neuroelectric brain activity.

Abstract: The inhibitory effect of the root of Cyclea peltata Lam. on nephrolithiasis induced in rats by feeding with ethylene glycolated water (1%) for 35 days was summarized. Ethylene glycol administration led to oxalate stone formation, as indicated by its high level in urine. Complementary to this anion, the cation calcium level in urine was elevated. These two ions may have contributed to the formation of calcium oxalate stones. In addition to high serum potassium, a low serum magnesium level contributed to stone formation. Simultaneous administration of the powdered root of Cyclea peltata resulted in decreased urinary oxalate and calcium. Likewise, serum potassium was lowered and magnesium was elevated. These observations provided the basis for the conclusion that this plant inhibits the stone formation induced by ethylene glycol treatment.

P-glycoprotein-mediated efflux as a major factor in the variance of absorption and distribution of drugs: modulation of chemotherapy resistance.

Abstract: Active efflux of many therapeutics and other xenobiotics from cells and tissues by P-glycoprotein (P-gp) can cause dramatic effects on bioavailability. This expulsion of compounds from cells is known as a major form of multiple drug resistance (MDR). Often a significant barrier to oral absorption at the intestine, P-gp also protects the liver, brain, placenta, testes, adrenal gland and other tissues from cytotoxic insult. Due to the wide tolerance of substrate recognition, P-gp can often be the mechanism for significant pharmacokinetic drug interactions when two or more drugs are competing for the P-gp transport site. P-gp levels are also inducible and can be even further elavated in cancer cells, thus contributing to the confounding pleiotropic resistance to chemotherapy and poor treatment prognosis. Consequently, a broad scope of research over 20 years has led to the evaluation of co-therapies intended to augment chemotherapy by inhibiting P-gp. This review includes a list of the currently known P-gp inhibiting adjuvant candidates described in the literature, with associated references and summary data. The summary catalogue of P-gp modulators illustrates the ardent pursuit to overcome this form of therapy resistance and gives examples of clinical success and failure.

Radioprotective effects of exogenous glutathione against whole-body gamma-ray irradiation: age- and gender-related changes in malondialdehyde levels, superoxide dismutase and catalase activities in rat liver.

Abstract: Age- and gender-related changes in malondialdehyde (MDA) levels, superoxide dismutase (SOD) and catalase (CAT) activities in rat livers exposed to different doses of whole-body gamma-ray radiation were determined. In addition, the effects of exogenous glutathione (GSH) against radiation injury in rat livers were investigated. We found that MDA levels have an age-associated increment and an increasing radiation dose-related elevation, although they decrease slightly in the 4 Gy group. The MDA levels in old rats were lower in males than in females, while those of young rats did not change. There were no observed age-related changes in SOD activities, although male rats had higher SOD activity than females. Female rats had the highest CAT activities in the 4 Gy group, while male rats had the highest CAT activities in the 6 Gy group. CAT activities in the 8 Gy group were lower than those of the 2 Gy group for each gender and age. While MDA levels were decreased and CAT activities increased by GSH, SOD activities remained unchanged. The results indicate that gamma-ray radiation affects gender- and age-dependent MDA levels, SOD and CAT activities. Administration of GSH appears to be a useful approach to reduce radiation injury by reducing MDA levels and increasing CAT activities.

Perceptual and cognitive event-related potentials in neuropsychopharmacology: methodological aspects and clinical applications (pharmaco-ERP topography and tomography).

Abstract: Middle latency and late components of event-related brain potential (ERPs) are closely related to perceptual and cognitive information processing, respectively. In a double-blind, placebo-controlled study, the acute effects of lorazepam (2 mg), haloperidol (3 mg), methylphenidate (20 mg) and citalopram (20 mg) on ERP latencies, amplitudes, topographies and tomographies were investigated in 20 healthy subjects of 23-34 years of age. After automatic artifact minimization and rejection, standard N1 and P2 and target N2 and P300 components were determined. The tranquilizer lorazepam prolonged P300 latency, which indicates an impairment of stimulus evaluation time. Low-resolution brain electromagnetic tomography (LORETA) revealed decreases in N1 and P300 source strength in those brain regions with relevant generators of these components, which reflects impairments of attentional and cognitive processing resources. The neuroleptic haloperidol decreased N1 and P300 source strength predominantly in those brain regions not involved in the generation of these components, suggesting a shift of resources. The psychostimulant methylphenidate increased P300 source strength in brain regions with major P3b generators, indicating increases in energetic resources associated with stimulus encoding. The antidepressant citalopram increased N1 and P3b source strength in multiple brain regions specifically in the left prefrontal cortex, a brain region in which reduced blood flow and metabolism was found in depressed patients.

Effect of melatonin and phenytoin on an intracortical ferric chloride model of posttraumatic seizures in rats.

Abstract: The present study was carried out to investigate the effect of melatonin, a potent antioxidant, and phenytoin, a conventional antiepileptic, against FeCl3-induced posttraumatic seizures. Male Wistar rats weighing 200-250 g were implanted with epidural electrodes and allowed to recover. After the recording of baseline EEG, FeCl3 (5 ul, 100 mM) was administered intracortically over a period of 5 min and EEG was monitored for 2 h. Subsequently, rats were sacrificed to estimate oxidative stress, i.e., the amount of malondialdehyde (MDA) in whole brain tissue. A sham group was run parallel with saline (pH adjusted), and a similar protocol for EEG recording and estimation of oxidative stress was followed. FeCl3-treated animals exhibited epileptiform EEG changes (high amplitude sharp waves of increased frequency and polyspikes) within 15 min, which continued throughout the period of observation. MDA levels were found to be significantly elevated as compared to the sham group. Melatonin (50 mg/kg i.p.) administered 30 min before FeCl3 injection delayed the onset of appearance of epileptiform EEG changes, while at a 100 mg/kg dose of there was complete protection, as none of the animal exhibited epileptiform EEG discharge. Brain MDA levels were also significantly reduced in melatonin (50 and 100 mg/kg dose)-treated animals as compared to the vehicle-treated FeCl3-injected rats. In the phenytoin group, all animals showed epileptiform EEG discharge. However, phenytoin at both 50 and 100 mg/kg dose delayed the onset of epileptiform EEG discharge. There were no significant changes in brain MDA levels in the phenytoin-treated group as compared to controls. Melatonin and phenytoin at doses of 100 mg/kg did not show any sign of motor impairment as observed during the rota-rod test. These findings showed a superior protective effect of melatonin over phenytoin in an intracortical FeCl3 model of posttraumatic epilepsy.

Combination of magneto- and electroencephalography in studies of monoamine modulation on attention.

Abstract: The brain monoamines serotonin and dopamine have an important role in the regulation of human cognitive functions. Neural correlates of attention can be studied in millisecond resolution with magnetoencephalography (MEG) and electroencephalography (EEC), which provide complementary views on attentional processing. During selective attention. processing negativity (PN) overlaps EEG response to the attended tones. This component can be modulated with drugs affecting dopamine transmission in the brain, such as haloperidol and droperidol, whereas no effects seem to be caused by acute tryptophan depletion (ATD), decreasing serotonin synthesis in the brain. Distinct responses are associated with involuntary attention. Responses reflecting sound-change detection and initiation of involuntary attention, the mismatch negativity and its magnetic counterpart. appear to be modulated by ATD, but not with haloperidol. Subsequent P3a elicited by actual attention shifting, as well as reorienting negativity reflecting orienting back to a relevant task. are in turn decreased by haloperidol, but not affected by ATD or other serotonin modulators. Serotonin may affect the earliest preattentive phases on auditory processing, indexed by mid-latency magnetic responses and Nlm, hut haloperidol's effects on these parameters are insignificant. Taken together, serotonin and dopamine may have differential effects on attentional processing depending on time after stimulus presentation.

Uses of pharmaco-EEG and pharmacokinetic-pharmacodynamic modeling in the clinical scenario.

Abstract: In order to place pharmaco-EEG within the clinical context, the distinction between biomarkers, surrogate endpoints, clinical endpoints and clinical outcomes is introduced. State-of-the-art applications of pharmaco-EEG, together with pharmacokinetic-pharmacodynamic modeling in everyday clinical practice in anesthesiology (semilinear canonical correlation), psychiatry (discrimination between responders and nonresponders to pharmacological treatment using the test dose), neurology (antiepileptic field) and neurophysiology (first-order Markov model of sleep stage transitions) are discussed. The combination of both procedures, although successfully used during some drug development programs (opioids or benzodiazepines), is not widely applied in the clinical scenario where the central nervous system (CNS) is concerned. Much work is still need to develop fully the potentials that pharmaco-EEG together with pharmacokinetic-pharmacodynamic modeling could bring to therapeutics in neuroscience.

Quality of life (Spain and France): validation of the chronic venous insufficiency questionnaire (CIVIQ).

Abstract: A health-related quality of life (HRQOL) assessment is particularly necessary for patients with chronic venous insufficiency (CVI), as is an assessment of the objective signs (edema, dilated veins, ulceration and other lesions). A comprehensive 3-year research program was undertaken in France to construct and validate the Chronic Venous Insufficiency Questionnaire (CIVIQ), a questionnaire designed specifically to evaluate the quality of life for CVI sufferers. The next step was to implement a program of cross-cultural validation in several countries. Translation into Spanish was undertaken following international guidelines. Face validity was verified with a pilot test among 12 Spanish patients. Psychometric validation was performed in Spain (n = 476 patients with CVI). The Spanish CIVIQ demonstrated very good internal consistency, high reproducibility and responsiveness, as well as longitudinal clinical validity. It appears to be a valuable instrument for assessing improvement in patient quality of life in response to both therapy in clinical practice and clinical trials.

Antiinflammatory properties of Hypoxis hemerocallidea corm (African potato) extracts in rats.

Abstract: The effects of aqueous and methanolic extracts of Hypoxis hemerocallidea corm, locally known as 'African potato' in South Africa, were examined on rat paw edema induced by subplantar injections of fresh egg albumin (0.5 ml/kg). Acetyl salicylic acid (100 mg/kg p.o.) was used as the reference antiinflammatory agent for comparison. Both the aqueous and methanolic extracts of H. hemerocallidea corm (500 mg/kg p.o.) progressively reduced rat paw edema induced by the subplantar injections of fresh egg albumin. The methanolic extract produced relatively greater and more pronounced antiinflammatory effect than the aqueous extract in the experimental animal model used. However, the two extracts of African potato examined in this study were found to be less potent than acetyl salicylic acid (ASA) as an antiinflammatory agent.

Experimental approaches to study PPAR gamma agonists as antidiabetic drugs.

Abstract: Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors that are related to retinoid, steroid and thyroid hormone receptors. Since PPARs appear to be closely involved in the regulation of dietary fat storage and catabolism, they have been established as an important target for the treatment of type 2 diabetes and other disorders associated with the high intake of dietary fat. Thiazolidinediones (TZDs) were the first class of compounds to be identified as PPAR gamma-ligands, constituting a new class of antidiabetic drugs that have recently been introduced as therapeutic agents for the treatment of type 2 diabetes mellitus by acting as insulin sensitizers. These compounds improve insulin resistance by increasing cell sensitivity to insulin. In fact, PPAR gamma agonists increase peripheral insulin sensitivity by increasing the transcription of genes, which, in turn, increase glucose uptake, also improving insulin-stimulated glucose disposal in muscle. PPAR gamma agonists increase insulin signaling, reduce circulating levels of free fatty acids and stimulate adipocyte differentiation, thus favoring the formation of smaller, more insulin-sensitive adipocytes. TZDs have been proven effective in different experimental models to evaluate their effectiveness as an antidiabetic agent, and the involvement of PPAR gamma in the pharmacological effects of these compounds has been supported by studies showing an excellent correlation between the hypoglycemic action of these drugs and their affinity for PPAR gamma. Despite this evidence, the site of action and the molecular mechanism of TZDs remain unclear. The aim of the present article was to review and discuss the most relevant pharmacological studies performed in the search for establishing the mechanism of action of antidiabetic TZDs and related compounds acting as PPAR gamma agonists, as well as to summarize those representative experimental approaches currently used to evaluate PPAR gamma agonists as therapeutic agents for the treatment of insulin-resistant type 2 diabetes mellitus.

Studies on the antilithic effect of Rotula aquatica lour in male Wistar rats.

Abstract: The decoction of Rotula aquatic a lour was screened for antilithic activity in male Wistar rats and the results were summarized based on the ionic changes in both urine and serum. Nephrolithiasis was induced in rats by feeding them 3% glycolic acid mixed feed for 45 days, which resulted in high urinary calcium, oxalate and high serum potassium. Simultaneous treatment with the decoction reduced calcium and oxalate ion concentration in urine, confirming the stone inhibitory effect. Histopathological studies of kidney tissue samples further substantiated the findings. The decoction was found to be nontoxic over the 45-day treatment peroid.

The pyridoindole antioxidant stobadine attenuates albuminuria, enzymuria, kidney lipid peroxidation and matrix collagen cross-linking in streptozotocin-induced diabetic rats.

Abstract: The aim of the present study was to investigate the effect of dietary supplementation with the pyridoindole antioxidant stobadine on kidney status and function in streptozotocin-induced diabetic rats. Diabetic male Wistar rats were fed a standard diet for 32 weeks or a diet supplemented with stobadine (0.05% w/w). The diabetic state was characterized by significantly elevated plasma levels of glucose, HbA1c and urea, severe reduction of total body weight and relatively enlarged kidneys. Elevated levels of conjugated dienes were recorded in the diabetic kidney confirming the presence of oxidative stress in diabetic animals. All diabetic rats showed marked proteinuria and albuminuria along with elevated excretion of the enzyme N-acetyl-beta-D-glucosaminidase. Long-term treatment of diabetic animals with stobadine significantly reduced total proteinuria, albuminuria and enzymuria, yet left the overall physical and glycemic status unaffected. It reduced oxidative damage of kidney tissue as shown by decreased conjugated diene level, and decreased matrix collagen cross-linking, as indicated by decreased breaking time values of rat tail tendons. These beneficial effects of stobadine, supported also by histological findings, may be brought about by virtue of the combination of its antioxidant potential with other effects, e.g., the postulated cholesterol-lowering ability or its ability to alter vascular reactivity and reduce the vascular tone.

Pharmacokinetic/pharmacodynamic relationships: basic concepts.

Abstract: Cytokine-activated endothelial expression of adhesion molecules plays an important role in immune responses. In the present study, we investigated the influences of testosterone and 17 beta-estradiol on tumor necrosis factor-alpha (TNF-alpha)-induced expression of adhesion molecules in human umbilical vein endothelial cells (HUVEC). HUVEC were incubated with TNF-alpha, testosterone or 17 beta-estradiol separately, or in a combination of TNF-alpha plus testosterone or 17 beta-estradiol. The expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) was evaluated at 3, 6, 12 and 24 h following exposure by flow cytometric analysis. The results showed that although testosterone or 17 beta-estradiol did not affect the expression of these adhesion molecules in unstimulated HUVEC, both of them transiently increased the expression of E-selectin and VCAM-1 in TNF-alpha stimulated HUVEC. Neither testosterone nor 17 beta-estradiol affected the expression of ICAM-1 induced by TNF-alpha. It is concluded that both testosterone and 17 beta-estradiol increase TNF-alpha-induced expression of E-selectin and VCAM-1 in endothelial cells and these facts might indicate a mechanism by which gonadal hormones can indirectly enhance immune responses.

Cholinergic function and dysfunction in the visual system.

Abstract: Acetylcholine (ACh) function is thought not only to play a significant role in memory, learning and other cognitive processes, but studies at a cellular level and in vivo indicate an important role for ACh in vision as well, especially for visual information processing. A suitable experimental model of geriatric memory impairment and Alzheimer dementia that pharmacologically blocks the brain muscarinic transmission has been proposed. This model has been extensively used also as an attempt to test cholinergic drugs in the absence of detailed knowledge of sites and mechanisms of ACh action and as test condition in the investigation of the role of ACh in visual information processing. Alzheimer's dementia results from complex neuron alterations, rather than simply reflecting ACh impoverishment, also involving the visual system, with substantial loss of retinal ganglion cells and alterations in visual information processing. Viewing all these data as a whole, nonspecific ACh actions on cognition, such as arousal or attention, contribute in modulating the function-specific action of ACh in information processing, both at cognitive and visual level.

Planning of pharmacodynamic trials: I. Specificity and possible solutions. II. Stability considerations under placebo and interpretation of drug effects on EEG.

Abstract: This workshop deals with the concept of quantitative electroencephalography (QEEG) to characterize the central effects of drugs. For proper interpretation, the circumstances under which data are obtained play an important role. To infer the size of variability in standard practice, we elaborated some computations for "placebo-treatment" in healthy volunteers, which helps to determine the threshold of drug effect detection. Simple rules for interpretation of multiple statistical comparisons were proposed and validation of dose effects were carried out with accepted reference compounds. Furthermore, psychotropic agents with comparable therapeutic indications often present similar modifications in EEG spectral composition (pharmaco-EEG profile). To extrapolate this concept, quantified wake-EEG is a rapid, validated technique for early psychopharmacological investigation of new psychotropic compounds (Phase I, healthy volunteers). Classification of drug-induced changes in cerebral activity at this stage forms a useful decision instrument in planning the long-term clinical scenario of drug development (Phases II and higher).

One-step reverse transcription polymerase chain reaction for semiquantitative analysis of mRNA expression.

Abstract: Analysis of mRNA expression is one of the main targets of scientific research. However, its quantification can be difficult, especially when dealing with low-expression mRNAs (ionic channels, carriers, receptors, etc.) or when only small samples are available (human biopsies). Here we suggest an easy, rapid and reliable method to assess semiquantitative changes in mRNA that combines several technical improvements: i) one-step reverse transcription polymerase chain reaction (RT-PCR) from total RNA; ii) addition of ethidium bromide to the gel, which provides a more homogeneous binding to DNA; iii) direct capture of the gel image using a charged-coupled device camera and then saving the image on computer before quantification, which increases resolution and thus improves and shortens the analysis; and iv) the use of 18S rRNA as a control, which is especially useful when samples from activation, differentiation and proliferation models are used. The technique was validated by checking the system conditions of image capturing and quantification. This was corroborated by a study of Kvl.3 ion channel expression in the brain. In these conditions, the wide range of PCR cycles and total RNA allows us to correlate relative gene expression and direct input of the target gene.

Recording EEG and EP data.

Abstract: Recording evoked potentials (EPs), electroencephalogram (EEG) signals and polysomnographic biosignals requires particular attention with respect to amplification, filtering, and data handling. The present paper is aimed at providing a short overview of the fundamentals on recording techniques and the basics on biosignal processing methods used in EEG studies. It also examines the aspects associated with the reproducibility of EEG measurements, as well as the recording duration by Pharmaco-EEG (PEEG) studies.

Basics of PK-PD using QEEG: acute/repetitive administration, interactions. Focus on anxiolytics with different neurochemical mechanisms as examples.

Abstract: Human Toll-like receptor 4 (TLR4) has recently been identified and has been shown to be the main protein involved in recognizing Gram-negative bacteria. We examined the regulation of TLR4 surface expression in a human monocytic cell line (THP-1 cells) by two traditional Chinese herbal medicines. Bu-Zhong-Yi-Qi-Tang (TJ-41) and Shi-Quan-Da-Bu-Tang (TJ-48). TJ-41 and TJ-48 upregulated TLR4 surface expression in THP-1 cells, as well as enhanced TLR4 surface expression in these cells both dose- and time-dependently. These findings suggest that TJ-41 and TJ-48 increase the receptor involved in the response to Gram-negative bacteria and may enhance defenses against these pathogens.