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Excitatory amino acid transporters (EAATs) remove glutamate from synapses. They maintain an efficient synaptic transmission and prevent glutamate from reaching neurotoxic levels. Glutamate transporters couple the uptake of one glutamate to the cotransport of three sodium ions and one proton and the countertransport of one potassium ion. The molecular mechanism for this coupled uptake of glutamate and its co- and counter-transported ions is not known. In a crystal structure of the bacterial glutamate transporter homolog, GltPh, only two cations are bound to the transporter, and there is no indication of the location of the third sodium site. In experiments using voltage clamp fluorometry and simulations based on molecular dynamics combined with grand canonical Monte Carlo and free energy simulations performed on different isoforms of GltPh as well on a homology model of EAAT3, we sought to locate the third sodium-binding site in EAAT3. Both experiments and computer simulations suggest that T370 and N451 (T314 and N401 in GltPh) form part of the third sodium-binding site. Interestingly, the sodium bound at T370 forms part of the binding site for the amino acid substrate, perhaps explaining both the strict coupling of sodium transport to uptake of glutamate and the ion selectivity of the affinity for the transported amino acid in EAATs.

Voltage-activated proton (Hv) channels are essential components in the innate immune response. Hv channels are dimeric proteins with one proton permeation pathway per subunit. It is unknown how Hv channels are activated by voltage and whether there is any cooperation between subunits during voltage activation. Using cysteine accessibility measurements and voltage-clamp fluorometry, we show data consistent with the possibility that the fourth transmembrane segment S4 functions as the voltage sensor in Ciona intestinalis Hv channels. Unexpectedly, in a dimeric Hv channel, the S4 in both subunits must move to activate the two proton permeation pathways. In contrast, if Hv subunits are prevented from dimerizing, the movement of a single S4 is sufficient to activate the proton permeation pathway in a subunit. These results indicate strong cooperativity between subunits in dimeric Hv channels.

Parasitic protozoa are unable to synthesize purines de novo and must import preformed purine nucleobases or nucleosides from their hosts. Leishmania major expresses two purine nucleobase transporters, LmaNT3 and LmaNT4. Previous studies revealed that at neutral pH, LmaNT3 is a broad specificity, high affinity nucleobase transporter, whereas LmaNT4 mediates the uptake of only adenine. Because LmaNT4 is required for optimal viability of the amastigote stage of the parasite that lives within acidified phagolysomal vesicles of mammalian macrophages, the function of this permease was examined under acidic pH conditions. At acidic pH, LmaNT4 acquires the ability to transport adenine, hypoxanthine, guanine, and xanthine with Km values in the micromolar range, indicating that this transporter is activated at low pH. Thus, LmaNT4 is an acid-activated purine nucleobase transporter that functions optimally under the physiological conditions the parasite is exposed to in the macrophage phagolysosome. In contrast, LmaNT3 functions optimally at neutral pH. Two-electrode voltage clamp experiments performed on LmaNT3 and LmaNT4 expressed in Xenopus oocytes revealed substrate-induced inward directed currents at acidic pH, and application of substrates induced acidification of the oocyte cytosol. These observations imply that LmaNT3 and LmaNT4 are nucleobase/proton symporters.

Voltage-gated potassium channels are comprised of four subunits, and each subunit has a pore domain and a voltage-sensing domain (VSD). The four pore domains assemble to form one single central pore, and the four individual VSDs control the gate of the pore. Recently, a family of voltage-gated proton channels, such as H(V) or voltage sensor only protein (VSOP), was discovered that contain a single VSD but no pore domain. It has been assumed that VSOP channels are monomeric and contain a single VSD that functions as both the VSD and the pore domain. It remains unclear, however, how a protein that contains only a VSD and no pore domain can conduct ions. Using fluorescence measurements and immunoprecipitation techniques, we show here that VSOP channels are expressed as multimeric channels. Further, FRET experiments on constructs with covalently linked subunits show that VSOP channels are dimers. Truncation of the cytoplasmic regions of VSOP reduced the dimerization, suggesting that the dimerization is caused mainly by cytoplasmic protein-protein interactions. However, these N terminus- and C terminus-deleted channels displayed large proton currents. Therefore, we conclude that even though VSOP channels are expressed mainly as dimers in the cell membrane, single VSOP subunits could function independently as proton channels.

Glutamate transporters (excitatory amino acid transporter (EAATs)) are critical for normal excitatory signaling and maintaining subtoxic glutamate concentrations in mammalian central nervous system. Recently, a crystal structure for a homologous transporter in bacteria was reported. Still, little is understood regarding the mechanism of substrate uptake. In transmembrane domain 4, the mammalian EAATs contain a stretch of over 50 amino acids (4B-4C loop) that are absent in the bacterial protein. These residues have been suggested to be located in the large extracellular vestibule seen in the crystal structure. State-dependent trypsin-cleavage sites have been reported in this region, suggesting that the 4B-4C loop undergoes significant conformational changes. Here we employed substituted cysteine accessibility, voltage clamp fluorometry, and fluorescence resonance energy transfer on oocytes expressing mutant EAAT3 transporters to determine the location and functionality of the 4B-4C loop. We find that this loop extends from near the center of the protein and that the majority of the residues are positioned on the outer perimeter of the protein, rather than inside the vestibule. Our fluorescence resonance energy transfer measurements demonstrated that these residues do not undergo large scale motions during glutamate uptake. However, our voltage clamp fluorometry studies indicate that these residues report on Na(+) and glutamate binding-induced conformational changes, including a previously un-described voltage-independent component of Na(+) binding to the transporter. The finding that residues far from the glutamate-binding site report on several different types of binding events suggests that the series of small conformational changes that accomplish glutamate uptake extend throughout the transporter structure.

Excitatory amino acid transporters (EAATs) use sodium and potassium gradients to remove glutamate from the synapse and surrounding extracellular space, thereby sustaining efficient synaptic transmission and maintaining extracellular glutamate concentrations at subneurotoxic levels. In addition to sodium-driven glutamate uptake, EAATs also mediate a glutamate-activated chloride conductance via a channel-like mechanism. EAATs are trimeric proteins and are thought to comprise three identical subunits. Previous studies have shown that the sodium-driven uptake of glutamate occurs independently in each of the three subunits. In contrast, a recent study reports high Hill coefficients for the activation of EAAT anion currents by glutamate and suggests that the subunits function cooperatively in gating the chloride conductance. In the present work, we find that the Hill coefficient for the activation of the anion current by glutamate is approximately 1 in both EAAT3 and EAAT4. Furthermore, we also used fluorescent labeling and inactivation correlation on EAAT3 and EAAT4 to determine whether the glutamate-activated chloride conductance is gated independently or cooperatively by the transporters. We found that both glutamate uptake currents and glutamate-activated chloride currents are mediated independently by each subunit of an EAAT multimer. It has been suggested that EAAT subtypes with particularly large anion conductances can directly influence the excitability of presynaptic terminals in certain neurons. Thus, the finding that the anion conductance is gated independently, rather than cooperatively, is important because it significantly alters predictions of the influence that EAAT-mediated anion currents will have on synaptic transmission at low glutamate concentrations.

The excitatory amino acid transporters (EAATs) play key roles in the regulation of CNS L-glutamate, especially related to synthesis, signal termination, synaptic spillover, and excitotoxic protection. Inhibitors available to delineate EAAT pharmacology and function are essentially limited to those that non-selectively block all EAATs or those that exhibit a substantial preference for EAAT2. Thus, it is difficult to selectively study the other subtypes, particularly EAAT1 and EAAT3. Structure activity studies on a series of beta-substituted aspartate analogues identify L-beta-benzyl-aspartate (L-beta-BA) as among the first blockers that potently and preferentially inhibits the neuronal EAAT3 subtype. Kinetic analysis of D-[(3)H]aspartate uptake into C17.2 cells expressing the hEAATs demonstrate that L-beta-threo-BA is the more potent diastereomer, acts competitively, and exhibits a 10-fold preference for EAAT3 compared to EAAT1 and EAAT2. Electrophysiological recordings of EAAT-mediated currents in Xenopus oocytes identify L-beta-BA as a non-substrate inhibitor. Analyzing L-beta-threo-BA within the context of a novel EAAT2 pharmacophore model suggests: (1) a highly conserved positioning of the electrostatic carboxyl and amino groups; (2) nearby regions that accommodate select structural modifications (cyclopropyl rings, methyl groups, oxygen atoms); and (3) a unique region L-beta-threo-BA occupied by the benzyl moieties of L-TBOA, L-beta-threo-BA and related analogues. It is plausible that the preference of L-beta-threo-BA and L-TBOA for EAAT3 and EAAT2, respectively, could reside in the latter two pharmacophore regions.

The dehydrogenation reaction of methanol on a Rh(111) surface, a Rh(111)V subsurface alloy, and on a Rh(111)V islands surface has been studied by thermal-desorption spectroscopy, reflection absorption infrared spectroscopy, and density-functional theory calculations. The full monolayer of methanol forms a structure with a special geometry with methanol rows, where two neighboring molecules have different oxygen-rhodium distances. They are close enough to form a H-bonded bilayer structure, with such a configuration, where every second methanol C-O bond is perpendicular to the surface on both Rh(111) and on the Rh(111)V subsurface alloy. The Rh(111)V subsurface alloy is slightly more reactive than the Rh(111) surface which is due to the changes in the electronic structure of the surface leading to slightly different methanol species on the surface. The Rh(111)V islands surface is the most reactive surface which is due to a new reaction mechanism that involves a methanol species stabilized up to about 245 K, partial opening of the methanol C-O bond, and dissociation of the product carbon monoxide. The latter two reactions also lead to a deactivation of the Rh(111)V islands surface.

Glutamate transporters remove glutamate from the synaptic cleft to maintain efficient synaptic communication between neurons and to prevent glutamate concentrations from reaching neurotoxic levels. Glutamate transporters play an important role in ischemic neuronal death during stroke and have been implicated in epilepsy and amytropic lateral sclerosis. However, the molecular structure and the glutamate-uptake mechanism of these transporters are not well understood. The most recent models of glutamate transporters have three or five subunits, each with eight transmembrane domains, and one or two membrane-inserted loops. Here, using fluorescence resonance energy transfer (FRET) analysis, we have determined the relative position of the extracellular regions of these domains. Our results are consistent with a trimeric glutamate transporter with a large (>45 A) extracellular vestibule. In contrast to other transport proteins, our FRET measurements indicate that there are no large-scale motions in glutamate transporters and that glutamate uptake is accompanied by relatively small motions around the glutamate-binding sites. The large extracellular vestibule and the small-scale conformational changes could contribute to the fast kinetics predicted for glutamate transporters. Furthermore, we show that, despite the multimeric nature of glutamate transporters, the subunits function independently.

Aims: In patients with persistent atrial fibrillation (AF), the efficacy and safety of two anti-arrhythmic drugs in preventing the recurrence of AF after successful direct current (DC) cardioversion was prospectively assessed in a multi-centre double-blind, placebo-controlled, randomised trial using daily trans-telephonic monitoring., Methods and Results: 1182 patients with persistent AF were prospectively enrolled, 848 patients were successfully cardioverted and then randomised to either sotalol (383 patients), quinidine plus verapamil (377 patients) or placebo (88 patients). The primary outcome parameter was AF recurrence or death. All patients received an event recorder (Tele-ECG) and had to record and transmit via telephone at least one ECG per day during follow-up. The mean follow-up period was 266 days. A total of 191,103 Tele-ECGs were recorded and transmitted. The primary outcome parameter (AF recurrence of any kind or death) was observed in 572 patients (67%) in whom at least one episode of AF recurrence was documented during follow-up, in 348 patients (41%) AF recurrence was persistent. The recurrence rates after one year for any AF were 83% for placebo, 67% for sotalol and 65% for quinidine plus verapamil, the latter being statistically superior to placebo but not different from sotalol. The recurrence rates for the secondary outcome parameter persistent AF were 77%, 49% and 38%, respectively. Quinidine plus verapamil was significantly superior to placebo and to sotalol. About 95% of all AF recurrences were initially detected in the daily Tele-ECG, about 70% of all AF recurrences occurred completely asymptomatic. Adverse events on sotalol and quinidine plus verapamil were comparable with the exception that all torsade de pointes tachycardias occurred on sotalol., Conclusion: Anti-arrhythmic treatment after DC cardioversion of persistent AF significantly decreases the recurrence rates of persistent AF compared to placebo with superiority of quinidine plus verapamil compared to sotalol. Symptoms were not reliable as clinical surrogates to detect episodes of AF.

Glutamate transporters remove glutamate from the synaptic cleft to maintain efficient synaptic communication between neurons and to prevent extracellular glutamate concentrations from reaching neurotoxic levels (1). It is thought that glutamate transporters mediate glutamate transport through a reaction cycle with conformational changes between the two major access states that alternatively expose glutamate-binding sites to the extracellular or to the intracellular solution. However, there is no direct real-time evidence for the conformational changes predicted to occur during the transport cycle. In the present study, we used voltage-clamp fluorometry to measure conformational changes in the neuronal excitatory amino acid transporter (EAAT) 3 glutamate transporter covalently labeled with a fluorescent reporter group. Alterations in glutamate and cotransported ion concentrations or in the membrane voltage induced changes in the fluorescence that allowed detection of conformational rearrangements occurring during forward and reverse transport. In addition to the transition between the two major access states, our results show that there are significant Na(+)-dependent conformational changes preceding glutamate binding. We furthermore show that Na(+) and H(+) are cotransported with glutamate in the forward part of the transport cycle. The data further suggest that an increase in proton concentrations slows the reverse transport of glutamate, which may play a neuro-protective role during ischemia.

Within the mammalian central nervous system, the efficient removal of L-glutamate from the extracellular space by excitatory amino acid transporters (EAATs) has been postulated to contribute to signal termination, the recycling of transmitter, and the maintenance of L-glutamate at concentrations below those that are excitotoxic. The development of potent and selective inhibitors of the EAATs has contributed greatly to the understanding of the functional roles of these transporters. In the present study, we use a library of conformationally constrained glutamate analogs to address two key issues: the differentiation of substrates from nontransportable inhibitors and the comparison of the pharmacological profile of synaptosomal uptake with those of the individual EAAT clones. We demonstrate that the process of transporter-mediated heteroexchange can be exploited in synaptosomes to rapidly distinguish transportable from nontransportable inhibitors. Using this approach, we demonstrate that 2,4-methanopyrrolidine-2,4-dicarboxylate, cis-1-aminocyclobutane-1,3-dicarboxylate, and L-trans-2, 4-pyrrolidine dicarboxylate act as substrates for the rat forebrain synaptosomal glutamate uptake system. In contrast, L-anti-endo-3, 4-methanopyrrolidine-3,4-dicarboxylate, L-trans-2,3-pyrrolidine dicarboxylate, and dihydrokainate proved to be competitive inhibitors of D-[(3)H]aspartate uptake that exhibited little or no activity as substrates. When these same compounds were characterized for substrate activity by recording currents in voltage-clamped Xenopus laevis oocytes expressing the human transporter clones EAAT1, EAAT2, or EAAT3, it was found that the pharmacological profile of the synaptosomal system exhibited the greatest similarity with the EAAT2 subtype, a transporter believed to be expressed primarily on glial cells.

Na+-dependent, high-affinity glutamate transporters in the central nervous system are generally credited with regulating extracellular levels of L-glutamate and maintaining concentrations below those that would induce excitotoxic injury. Under pathological conditions, however, it has been suggested that these same transporters may contribute to excitotoxic injury by serving as sites of efflux for cellular L-glutamate. In this study, we examine the efflux of [3H]D-aspartate from synaptosomes in response to both alternative substrates (i.e., heteroexchange), such as L-glutamate, and a metabolic insult (5 mM potassium cyanide and 1 mM iodoacetate). Exposure of synaptosomes containing [3H]D-aspartate to either L-glutamate or metabolic inhibitors increased the efflux of the radiolabeled substrate to over 200% of control values. Two previously identified competitive transport inhibitors (L-trans-2, 3-pyrrolidine dicarboxylate and dihydrokainate) failed to stimulate [3H]D-aspartate efflux but did inhibit glutamate-mediated heteroexchange, consistent with the action of nontransportable inhibitors. These compounds also attenuated the efflux of [3H]D-aspartate from synaptosomes exposed to the metabolic inhibitors. These results add further strength to the model of central nervous system injury-induced efflux of L-glutamate through its high-affinity transporters and identify a novel strategy to attenuate this process.

Atrial fibrillation (AF) is the most frequent cardiac arrhythmia. However, despite manifold publications reflecting numerous clinical trials about treatment of AF, the management of this arrhythmia is still under controversial discussion, in daily clinical work as well as in research. The present study concentrates on three major questions: 1. How frequent are recurrences of AF in long-term follow-up? Most of the previous studies used the occurrence of symptoms as a surrogate parameter for recurrences of AF, despite the expected high rate of asymptomatic relapses. In the present study a daily transtelephonic ECG transmission enables a rhythm monitoring independent of symptoms. 2. Is the frequency of AF recurrences significantly reduced by antiarrhythmic medication? A direct comparison of class I and III antiarrhythmic drugs, which still are most frequently used for this indication, and of placebo will answer this question. 3. How safe is the long-term treatment for the prevention of AF recurrences with special respect to proarrhythmic effects? The daily transtelephonic ECG transmission enables a quantitative and qualitative monitoring of tachy- and bradyarrhythmias independent of symptoms. Additionally, the daily analysis of ECG measures may detect parameters predicting subsequent life threatening arrhythmias. The study design provides a prospective, randomised, double-blind, placebo controlled, multicenter parallel group comparison. In Germany and in the Czech Republic about 90 hospitals will include 900 patients with documented chronic AF, age 18 to 80 years, if they are eligible for electrical cardioversion without concomitant antiarrhythmic drug therapy and if they are anticoagulated for at least three weeks prior to inclusion. Neither the size of the left atrium nor the duration of chronic AF are exclusion criteria. A few hours after successful electrical cardioversion the patients are randomised either to sotalol (2 x 160 mg) or quinidine + verapamil (3 x 160 mg + 3 x 80 mg) or placebo. Starting at the day after cardioversion, the patient is asked to record and transmit electrocardiograms of one minute duration at least once a day using his personal transtelephonic ECG recording unit (Tele-ECG recorder, credit card size), in case of symptoms as often as necessary. The ECGs can be transmitted at any time by any regular phone without additional equipment using a toll free number. A custom made, computer based, fully automated receiving centre is handling the patient calls interactively with voice control, including a voice recording of the patient's symptoms. The ECG tracings and the patient's voice messages are subsequently computer based analysed by experienced technicians. All ECG measures are stored in a database. In case of AF recurrence, any other relevant arrhythmia or additional abnormalities (e.g. QT prolongation) the correspondent hospital is immediately informed by fax. In case of AF recurrence, a subsequent Holter recording discriminates in paroxysmal and permanent AF. Study medication is ended if either permanent AF or the third episode of paroxysmal AF are detected or after 12 months of follow-up. Regular follow-up visits are performed monthly. Major endpoints are the time to first recurrence of AF or the time to death, secondary parameters are the number of AF recurrences, the time to end of medication and AF related symptoms. The recruitment started in the last days of 1996. Until the end of June 1998, 424 patients have been randomised. It is expected to end recruitment in spring 1999 and to close the study in spring 2000. Final results will be available in summer 2000.

The indication to treat symptomatic paroxysmal atrial fibrillation is discussed controversely. Successful medical treatment may result in the reduction of symptoms by improving hemodynamics in a reduction of thromboembolic events. However, several antiarrhythmic drugs are also known to increase the risk of proarrhythmic events. A randomized, double-blind, and placebo-controlled multicenter trial with 1000 patients to be recruited was designed to compare the effects of two antiarrhythmic drugs frequently used in Germany for the treatment of atrial fibrillation, Sotalol and the fixed combination of chinidin and verapamil (Cordichin). Patients with symptomatic paroxysmal atrial fibrillation/atrial flutter will be observed for a period of one year. The occurrence of paroxysmal atrial fibrillation is documented by transtelephonic ECG monitoring. Patients with document an ECG once daily, and recording is mandatory in case of symptoms. ECGs are transmitted to a central data base for analysis. This clinical trial is designed to answer the following questions: (1) What is the average rate of spontaneous events of symptomatic atrial fibrillation? (2) Is it possible to reduce the frequency of symptomatic events by chronic antiarrhythmic drug administration? (3) What is the long-term frequency for the occurrence of severe side-effects under antiarrhythmic medication? The primary endpoint is defined as the time to first recurrence of symptomatic arrhythmia after reaching steady-state plasma concentrations of the study medication. The trial started in November 1997 and is planned to be finished by the end of 1999.

Using an intramolecular [2 + 2] photocyclization, 2,4-methanopyrrolidine-2,4-dicarboxylate was prepared as a conformationally locked analogue of glutamate. This compound, in combination with two other pyrrolidine dicarboxylates, has been used to define the structural elements that differentiate substrate and nonsubstrate inhibitors of a high-affinity, sodium-dependent glutamate transporter.

This study investigated the in vitro and in vivo excitotoxic properties of a novel conformationally constrained analogue of L-glutamate, L-trans-2,3-pyrrolidine dicarboxylate (L-trans-2,3-PDC). When tested for excitotoxic activity in rat cortical cultures, L-trans-2,3-PDC mimicked the action of NMDA in both acute (30 min) and chronic (24 h) exposure paradigms. This neurotoxicity was attenuated by co-addition of MK-801 (10 microM). Microinjections of L-trans-2,3-PDC into the dorsal hippocampus of male rats also induced a selective pattern of pathology indicative of an NMDA receptor excitotoxin. In contrast to the equipotency observed in vitro, 100 nmol of L-trans-2,3-PDC were needed to produce cellular damage comparable to that induced by 25 nmol of NMDA. Consistent with an action at NMDA receptors, L-trans-2,3-PDC-induced damage could be significantly reduced by co-administration of MK-801 (3 mg/kg i.p.), but not by NBQX (25 nmol). In radioligand binding assays L-trans-2,3-PDC inhibited the binding of 3H-L-glutamate to NMDA receptors (IC50 1 microM), although it also exhibited some cross reactivity with KA and AMPA receptors. L-trans-2,3-PDC was also identified as a competitive inhibitor (Ki = 33 microM) of 3H-D-aspartate uptake into rat forebrain synaptosomes. In contrast to the action of a transported substrate, such as L-glutamate, L-trans-2,3-PDC did not exchange with 3H-D-aspartate that had been previously loaded into the synaptosomes.

An old problem is sometimes better understood when it is viewed from a new perspective than by investigating it repeatedly with classical methods. Such a new perspective is provided by the concept of fractals. Briefly, this term defines a spread class of geometric shapes, whose subunits replicate the structure of the larger unit in accordance with the formalization of the idea of self-similarity. This concept was introduced by Mandel-brot in respond to the need for a more sophisticated explanation of numerous phenomena commonly encountered in nature. In this review we will provide a short introduction into the concept of fractals and a concise overview of those studies where fractal geometry has been employed so far in pharmaceutical research. We believe that a review of the most recent findings in the application of fractal geometry to problems encountered in the pharmaceutical sciences might be helpful also to those people who are involved in the search for novel drug substances, as well as in the investigation of their performance in vitro and in vivo. A few suggestions will be made where this concept probably could be helpful in the future and instructions will be given on how the fractal dimension of rugged objects can be estimated practically.

The pharmacokinetics and organ distribution of 3H-L-carnitine (CAS 541-15-1) were investigated in rats following direct intravenous administration of the drug substance and administration of the drug encapsulated in liposomes, respectively. The retention in the blood system of carnitine in liposomes, was significantly higher, namely up to 300% as compared to the standard administration. The half-life of distribution t1/2 alpha to 0.68 h (+154%), the terminal half-life t1/2 beta to 7.94 h (+140%), whereas the total clearance decreased by 400% as compared with the standard carnitine administration. Carnitine, in the novel dosage form, accumulated to a higher extent in the liver (156%) and spleen (336%), while the concentration in lung (52%), heart (55%) and muscle tissue (54%) decreased markedly relative to the standard. The novel dosage form is stable in vitro (t1/2 4 degree C = 187 days) as well as in vivo and, thus, may be successfully used in the therapy of carnitine deficiency, for instance in patients with renal failure or liver disease.

A novel testing procedure has been developed with the aim to replace the traditional LD50 test in vertebrates by a method using a non-pain sensitive organism. Several years of practical experience have proven this method to be a rather quick, simple, inexpensive, outstandingly well reproducible and reliable experimental technique which yields an estimate for the acute toxicity of drugs, environmental chemicals, solvents, food additives, pesticides, industrial and waste products, and the like. The model is equivalent to the customary LD50 test in mice, rats and other laboratory animals. The yeast test, as it has been briefly named, employs ordinary yeast (Saccharomyces cerevisiae) in a thermostatized incubation mixture with nutrients and trace elements. The test substance is added to this mixture by increasing concentration, and the effect upon the growth rate of the yeast cells is monitored at 30, 90, 150 and 210 min after beginning the experiment by counting the cell number, either in a simple counting chamber under the microscope or, more conveniently, by using an electronic Coulter counter. The effect is expressed as percent growth of the cells in relation to the untreated control. Evaluation of the experimental data leads to a general toxicity parameter, the mean inhibitory concentration or IC50 value of the compound under test. Hitherto it was found that the IC50 values of approx. 160 common drugs and other chemicals correlate well with the known LD50 values found in animals with the same substances.

To assess the antiischemic efficacy of slow-release (SR) gallopamil, 100 mg b.i.d., versus slow-release (SR) nifedipine, 20 mg b.i.d., 24 patients with chronic stable angina underwent symptom-limited bicycle ergometer exercise stress tests in a randomized, placebo-controlled, double-blind, cross-over protocol. Both medications caused a significant reduction in anginal attack frequency and nitroglycerin consumption as compared to placebo; similarly, exercise tolerance was augmented in association with a considerable reduction in ischemia-induced ST-segment depression. The antiischemic effect of gallopamil (SR) was marginally superior to that of nifedipine (SR). Since the incidence of adverse effects was also less with gallopamil (SR) this drug exhibited a more favorable risk-benefit ratio relative to nifedipine (SR).

The effects of 20 flavonoids, anthocyanes and other phenolic compounds of plant origin have been tested in vitro for their inhibitory effect on the enzyme adenosine deaminase (ADA). Significant effects were obtained with most compounds at concentrations between 0.0001 and 0.1 mg/mI. Thus, plant phenolics can be considered as natural ADA enzyme inhibitors. It is assumed that they also increase the actual adenosine (Ado) concentration in vivo. This may explain many of the pharmacological activities of these compounds on a molecular basis.

Starting point for this study was the urgent need for replacement of the contemporary mode of acute toxicity testing of chemicals in vertebrates (LD50-test) by an experimental model with equal power that can be performed on non-pain sensitive matter. For this purpose, a testing procedure ought to be developed using a non-pathogenic microorganism as testing object that is available at any time, easy to cultivate, and in its indicative power equivalent to the LD50 test in mice, rats, and other laboratory animals. Such an organism has been found with ordinary yeast (baker's yeast, brewer's yeast, Saccharomyces cerevisiae) which can always be obtained in good quality. This procedure (shortly denominated as 'yeast test' offers several advantages: It is rather simple, inexpensive, outstandingly well reproducible, and it can be carried out conveniently also in routine experiments. The results correlate well with those furnished by the customary methods of acute toxicity testing.

A series of computer programs (BASIC) is introduced for the evaluation of in vitro dissolution data as well as for the calculation of pharmacokinetic parameters and for blood level curve fitting to the respective in vitro data. Furthermore, programs are offered for the simulation of blood level profiles under variable conditions. In order to demonstrate the practical use of these programs, the expected blood level profiles following the administration of two commercial sustained-release dosage forms containing the drug pentoxifylline have been compared.

The anti-ischemic properties and tolerability of a slow-release formulation (SR) of gallopamil were investigated in 118 patients with exercise-inducible ST-segment depression and stable angina pectoris in this double-blind, randomized, placebo-controlled, multicenter study. After a placebo run-in period (A) of 2-7 days and a 7-day open therapy period (B) with gallopamil SR, the patients were randomized to a double-blind 7-day period (C) to receive placebo or gallopamil SR 100 mg twice a day. Each patient was submitted to gradual upright bicycle ergometry and electrocardiography (ECG) at rest on the last 2 days of each period at 6 and 12 h postadministration (p.a.) In period C, exercise time and exercise tolerance remained significantly prolonged at 6 and 12 h after gallopamil SR administration in comparison with the placebo values. Additionally the sum of ST-segment depression and maximal ST-segment depression were significantly reduced by gallopamil SR at 6 h p.a. as were the frequency of angina attacks and nitroglycerin consumption. Four patients were withdrawn from the study because of gallopamil-related adverse events, which, however, were not serious. Constipation was noted in 2.5% of the patients. These data suggest that gallopamil SR is effective in reducing exercise-inducible ST-segment depression and increasing exercise tolerance with no serious adverse effects in patients with stable angina pectoris.

Under certain circumstances the time course of drugs and/or metabolites in body fluids and tissues may show a 'fractal pattern', i.e. manifestations of mainly irregular figures having identical or similar structures in their parts as in the whole. This will be demonstrated in this paper by means of just one example, although many more can be found easily in the literature. It appears that the fractal concept may also be useful in pharmacokinetics to describe the complexity of time-concentration curves by an additional parameter, the 'fractal dimension'. A simple principle is proposed for testing the 'self-similarity' which is useful as accompanying evidence of the fractal dimension.

This report summarises selected preliminary results from an ongoing study designed to investigate the effect of the calcium antagonist gallopamil on myocardial ischaemia during percutaneous transluminal coronary angioplasty (PTCA). To date, 12 adult males with coronary artery disease and significant proximal stenosis of the left anterior descending coronary artery (LAD) have been randomly assigned to gallopamil or placebo under double-blind conditions. Patients with recent myocardial infarction, apparent collateralisation of the LAD, myocardial failure, sinoatrial or atrioventricular block, severe hepatic disease or renal failure were excluded from the study. PTCA was performed using at least 2 balloon inflations, each of 2 minutes' duration. Gallopamil 0.4 mg or placebo (normal saline) were administered during the 10-minute interval between the 2 inflations. Blood samples were taken simultaneously from the coronary sinus and the femoral artery before and immediately after each inflation. Lactate concentration and the relative amount of activated neutrophils were selected for trend analysis. Furthermore, ECG changes were analysed by calculating the sum of the absolute ST-segment deviations (80 msec after J point, maximal T deviation) of leads I, II, III, V2, V4 and V6. In the presence of gallopamil, the degree of ST-segment/T-wave changes induced by balloon inflation was reduced. Additionally, gallopamil attenuated myocardial lactate release and appeared to prevent the increase in activated neutrophils observed during control inflations. These preliminary results suggest a beneficial effect from intracoronary administration of gallopamil during PTCA, achieved by attenuation of the ischaemic reaction during coronary occlusion.

Tritium-labelled beta-sitosterol (BSS) 10 mg was administered to 6 beagle dogs in a 3-way crossover study: 1. i.v. solution, 2. p.o. powdered BSS, and 3. BSS embedded in a polyethyleneglycol (PEG) melt. The concentration-time profiles for both routes of administration were best described by a two-compartment open model with a fast distribution phase, having a t1/2 alpha of about 3 h, and a terminal disposition phase having a t1/2 beta of about 129 h. The volume of distribution of the central compartment corresponds to the total body fluid (Vc = 0.56 l/kg), and the apparent volume of distribution is about equal to the total body weight (V d/beta = 0.92 l/kg). The mean residence time is about 185 h. The absolute bioavailability upon p.o. administration is about 9%. The PEG embedment of BSS does not increase the extent of absorption; however, the rate of absorption is significantly increased.

A total of 120 samples of serum were obtained from 8 patients with COPD and submitted to a comparative analysis of serum theophylline levels employing the TDX and the EMIT methods of determination. At the same time, two different slow-release preparations of theophylline were compared, their kinetics investigated, and their effects tested using body plethysmography. For this open, randomized cross-over preliminary investigation, numerous inclusion and exclusion criteria were applied.

The U.S.P./N.F. dissolution test (method I or rotating basket method) and the rotating flask technique (RESO-TEST dissolution method) were applied to four commercial prolonged release theophylline dosage forms. The dissolution data obtained were converted into dissolution efficiencies and submitted to a correlation analysis, the result of which demonstrates the equivalency of both methods with respect to the characterization of the dissolution behaviour of the dosage forms tested. It is suggested to consider replacement of the official dissolution technique, the shortcomings of which are well-known from the literature, by the rotating flask technique.

In this communication, a report is presented on the drug liberation from so-called floating capsules (with 10 and 15 mg diazepam) and from several other comparable oral and rectal drug dosage forms in the rotating flask model (Resotest-System). None of the commercially available preparations exhibits any availability problem. The rotating flask was suitable and appears as superior over the official dissolution apparatuses with regard to the testing of drug dosage forms that float on the surface of the dissolution medium.

The influence of two important factors, the rotational speed and the fluid volume within the system, on the release characteristics of prednisone-test tablets in the rotating flask dissolution-tester (Resotest-apparatus after Koch) has been investigated. As a result, standard conditions for dissolution experiments with the rotating flask model, i.e. a mean rotation number of 30 r.p.m. and an actual fluid volume of 500 ml, are proposed.

To study a possible drug interaction between cimetidine (C) and sucralfate (S) a crossover design was employed using four beagle dogs in each of the three groups: C alone, S alone, C plus S. A one week washout period elapsed between each treatment. C (300 mg, spiked with 3H material 9.9 microCi) was given P.O. by stomach catheter. S (1000 mg) was given as intact tablet. To group 3, C was administered 1 h after S dosing. In presence of S ka of C decreased significantly from 3.99 +/- 0.75 to 2.31 +/- 0.19 (SD) 1/h, and tmax increased significantly from 0.59 +/- 0.12 to 1.00 +/- 0.08 (SD) h. In presence of C Cmax and AUC (0-8) of aluminum increased from 21.38 +/- 4.78 to 29.25 +/- 5.86 (SD) mcg/ml and from 132.3 +/- 40.8 to 168.1 +/- 22.0 (SD) (mcg/ml) h. Absorption of C is delayed in the presence of S. Concomitant administration of C and S results in increased systemic availability of aluminum.

A rapid and sensitive isocratic high-performance liquid chromatography assay for the simultaneous and quantitative determination of thalidomide (alpha-phthalimidoglutarimide) and its major metabolites from human serum has been developed. The parent compound and the metabolites can be efficiently separated by reversed-phase chromatography using tetramethylammonium bromide as an ion-pair-forming reagent.

The pharmacokinetics of noreximide in the rat after i.v., p.o., and i.p. administration was studied. The biologic half-life of approximately 8 h was found almost the same for all three routes. Upon p.o. administration the fraction of drug absorbed is 85 per cent, and the peak concentration in the blood is reached within approximately 4.5 h. Addition pharmacokinetic parameters are listed.

The in vitro dissolution of four commercially available sustained-release theophylline products was studied. The dissolution of theophylline was evaluated under four conditions: USP-method I or Resotest apparatus, and artificial gastric (pH 3) or artificial intestinal (pH) fluids. The resulting data were evaluated by graphic, first-order transform, dissolution efficiency, non-linear fitting, mean time of dissolution and Weibull analysis methods. The four theophylline preparations and a peroral solution were administered to four male beagle dogs in a cross-over fashion. Parameters were obtained from graphed concentration vs. time data, a one-compartment open model fit, Wagner-Nelson absorption plots, ratios of half values of duration and maximum concentrations, mean times of absorption and analog computer evaluation. Correlations were made between all possible combinations of in vitro and in vivo data by quadrant analysis, Pearson product moment, r, and Spearman rank order correlation methods. A total of 8280 correlation coefficients were calculated. The optimal set of studied conditions for in vivo-in vitro correlations with sustained-release theophylline preparations is the time to reach maximum concentration in the blood (from the fitted data) vs. the time for 15% of the drug to be released into solution from the dosage form (from graphed data) using either the USP I apparatus or the Resotest.

The psychopharmacologically active drug noreximide was administered in a crossover design to beagle dogs intravenously, and perorally in form of a solution and as capsule. The dosage forms were prepared from 14C-labeled noreximide and cold material. The total radioactivity and that of the metabolite were determined in plasma after TLC separation. The mean half-life of the parent compound is about 4.5 hours after I.V. and 9.5 hours after P.O. administration. The apparent volume of distribution is about 20 l/kg, and the total clearance about 44 l/h after I.V. and about 29 l/h after P.O. dosing. The absolute bioavailability is about 50%. The drug is completely absorbed but undergoes extensive first-pass metabolism. The main metabolite, noreximide-epoxide has an elimination half-life of about 20 hours and an apparent volume of distribution identical to that of the parent compound. The peak metabolite concentrations occur about 10 hours after dosing.

Sixteen natural phenolics and semisynthetic derivatives thereof, including silymarin, flavonoids, catechines and phenolic acids, together with 6 standard drug substances with anti-inflammatory, antioxidant and peroxide radical scavenging properties have been tested in an in vitro model using human platelets for their inhibitory action against N-ethyl maleimide-induced lipid peroxidation. The malondialdehyde formed during the reaction was estimated by means of the thiobarbituric acid method. In all compounds tested, the inhibitory action was clearly dose-dependent; IC50 values were calculated from the dose-activity curves. The IC50 found ranged from between 1.47 nM and approximately 900 mM.

'Disintegration' and 'dissolution' of solid drug dosage forms are considered as partial processes of drug liberation. Besides the (apparent) disintegration time that is determined in the official disintegration tester, there is a second (intrinsic) disintegration time which can be deduced from the experimental dissolution data, but shows only limited correlation with the former. Various methods for the graphical resolution of dissolution data and the calculation of the intrinsic disintegration time therefrom are discussed. For the simultaneous determination of disintegration and dissolution, the approved rotating flask method is recommended.

Norendimide, the endo-isomer of the new psychoactive compound noreximide, was studied in rats upon I.V., P.O., and I.P. administration, and the pharmacokinetic parameters were determined from the concentration-time data. The one-compartment model was most appropriate for curve-fitting of all data. The elimination half-life is approximately 11 hours, and hence about 30% longer than that of the parent compound. The volume of distribution is about 85% of the body weight. Upon P.O. and I.P. administration the fraction of drug absorbed is 80% and 94%, respectively. A comparison with the previously reported pharmacokinetic data on noreximide indicates that both compounds, the parent drug and the analog, are completely absorbed and show some first-pass effect.