In vitro and in vivo P-glycoprotein transport characteristics of rivaroxaban

Rivaroxaban, an oral, direct factor Xa inhibitor, has a dual mode of elimination in humans, with two-thirds metabolized by the liver and one-third renally excreted unchanged. P-glycoprotein (P-gp) is known to be involved in the absorption, distribution, and excretion of drugs. To investigate whether rivaroxaban is a substrate of P-gp, the bidirectional flux of rivaroxaban across Caco-2, wild-type, and P-gp-overexpressing LLC-PK1 cells was investigated. Furthermore, the inhibitory effect of rivaroxaban toward P-gp was determined. Rivaroxaban exhibited high permeability and polarized transport across Caco-2 cells. Rivaroxaban was shown to be a substrate for, but not an inhibitor of, P-gp. Of a set of potential P-gp inhibitors, ketoconazole and ritonavir, but not clarithromycin or erythromycin, inhibited P-gp-mediated transport of rivaroxaban, with half-maximal inhibitory concentration values in the range of therapeutic plasma concentrations. These findings are in line with observed area under the plasma concentration-time curve increases in clinical drug-drug interaction studies indicating a possible involvement of P-gp in the distribution and excretion of rivaroxaban. In vivo studies in wild-type and P-gp double-knockout mice demonstrated that the impact of P-gp alone on the pharmacokinetics of rivaroxaban is minor. However, in P-gp double-knockout mice, a slight increase in brain concentrations and decreased excretion into the gastrointestinal tract were observed compared with wild-type mice. These studies also demonstrated that brain penetration of rivaroxaban is fairly low. In addition to P-gp, a further transport protein might be involved in the secretion of rivaroxaban.     

In vitro and in vivo transport of lithium by human erythrocytes.

An in vitro system that can be used to measure both uptake and efflux of lithium by erythrocytes (RBCs) is described. Using this system, RBC lithium accumulation in vitro was compared with in vivo RBC lithium concentrations observed in 6 normal volunteers. A significant correlation was demonstrated between in vitro RBC lithium accumulation after 48-hr incubation and in vivo RBC lithium concentration at 24, 48, 72, and 96 hr following the beginning of lithium ingestion. In addition, when efflux of lithium from RBCs in vitro was studied, a significant correlation was observed between residual lithium in RBCs and in vitro RBC lithium accumulation. Finally, it has been demonstrated that storage of blood in ice for 5 hr prior to incubation with lithium results in increased RBC lithium accumulation. A potential role for this in vitro incubation system as a model for in vivo RBC lithium accumulation is suggested.     

In vitro and in vivo studies on mucoadhesive microspheres of amoxicillin.

Amoxicillin mucoadhesive microspheres (Amo-ad-ms) were prepared using ethylcellulose (Ec) as matrix and carbopol 934P as mucoadhesive polymer for the potential use of treating gastric and duodenal ulcers, which were associated with Helicobacter pylori. The morphological characteristics of the mucoadhesive microspheres were studied under scanning electron microscope. In vitro release test showed that amoxicillin released faster in pH 1.0 hydrochloric acid (HCl) than in pH 7.8 phosphate buffer. Yet, it would be degraded to some extent in a pH 1.0 HCl medium at 37 degrees C, which indicated that amoxicillin was not stable in an acidic surrounding. It was also found that amoxicillin entrapped within the microspheres could keep stable. In vitro and in vivo mucoadhesive tests showed that Amo-ad-ms adhered more strongly to gastric mucous layer than nonadhesive amoxicillin microspheres (Amo-Ec-ms) did and could retain in gastrointestinal tract for an extended period of time. Amo-ad-ms and amoxicillin powder were orally administered to rats. The amoxicillin concentration in gastric tissue was higher in the Amo-ad-ms group. In vivo H. pylori clearance tests were also carried out by administering, respectively, Amo-ad-ms or amoxicillin powder, to H. pylori infectious BALB/c mice under fed conditions at single or multiple dose(s) in oral administration. The results showed that Amo-ad-ms had a better clearance effect than amoxicillin powder did. In conclusion, the prolonged gastrointestinal residence time and enhanced amoxicillin stability resulting from the mucoadhesive microspheres of amoxicillin might make contribution to H. pylori clearance.     

New echocardiographic and angiographic methods for right atrial volume determination: In vitro validation and in vivo results

Until now, right atrial (RA) volume calculation by means of two-dimensional echocardiography (2-DE) has only been attempted in a single plane: the apical four-chamber view. Our study reports a new method for RA volume calculation using two intersecting 2-DE views. For this purpose, silicone rubber casts of 19 human necropsy hearts were obtained and thin-walled natural rubber moulds of the RA casts were prepared. Totally filled with and immersed in water, the moulds could be visualized in the apical four-chamber view and an additional 2-DE plane, the latter corresponding to the subcostal view in vivo. In this view the vertical extension of RA could be estimated. Areas and lengths of RA were determined in the respective planes, and RA volume was calculated by applying the formula, area x length, to two intersecting planes. Finally, volume of the silicone casts was determined angiocardiographically (Angio) using a biplane method (30° RAO, 40° LAO-40° hepatoclavicular). The true RA volume was 106±23 ml (mean±1SD) as determined by water displacement. Using Angio an excellent correlation was found: the calculated volume amounted to 106±23ml; the difference was 5.5±4.8ml (n.s.); Angio vol=0.93 true vol+ 7.77; r=0.95; SEE= 7,4 ml. Volume determination from the apical four-chamber view of 2-DE using a monoplane disk method resulted in a mean volume of 62±17 ml. The mean difference to the true RA volume was 44±16 ml (p < 0.001). When volume calculations were made using the biplane method, a value of 105±22 ml resulted. The mean difference to true volumes was 7.4±4.8 ml: y=0.84x + 15.88; r=0.91; SEE=9.4 ml. In an in vivo study endsystolic RA volumes were calculated in a normal adult population (n=40) from the same intersecting planes as in vitro. A normal value of 38±6 ml/m² was found. In vivo validation using Angio showed a slightly higher normal value of 43=7 ml/m². Thus, 2-DE is highly accurate in determinating RA volume. In the in vitro as well as in the in vivo study the results of monoplane calculations are clearly inferior to a method which also takes account of the vertical extension of RA.     

In vitro and in vivo transport and delivery of phosphorothioate oligonucleotides with cationic liposomes

A recent strategy in gene therapy has been using antiviral genes that are delivered to uninfected cells, either as RNA or DNA, to provide intracellular protection from human immunodeficiency virus type-1 (HIV-1) infection. Antisense oligonucleotides that are complementary to specific target genes suppress gene expression. A variety of techniques are available to enhance the cellular uptake and pharmacological effectiveness of antisense oligonucleotides, both in vitro and in vivo. We investigated the intracellular and tissue uptake of an oligonucleotide/cationic lipid complex, using a fluorescently labeled oligonucleotide. The antisense oligonucleotide was designed against the HIV-1 gag gene sequence. A T-cell line (MT-4) and PHA-stimulated peripheral blood mononuclear cells (PBMCs) were both infected with HIV-1(NL432) at an MOI of 0.01. One h later, both cultures were washed and treated with medium containing 1 microM antisense oligonucleotide. After a 3-day interval, the HIV-1 antigen expression was monitored by an indirect immunofluorescence assay. At 3 days post infection, we confirmed that p24 antigen production was inhibited by the antisense oligonucleotide/cationic lipid complex at a 1/10 ratio in the PBMCs, using enzyme-linked immunosorbent assay (ELISA). We also confirmed the intracellular existence of the complex by fluorescent microscopy. We investigated different means of transporting the antisense oligonucleotide/cationic lipid complex to mouse tissues by intravenous, intraperitoneal and subcutaneous injections. We observed that the anti-HIV-1 activity of the antisense oligonucleotide/cationic lipid complex was the result of enhanced cellular uptake, both in vitro and in vivo. Therefore, the antisense oligonucleotide/cationic lipid complex is an excellent system for the transport and delivery of genes to target cells, as it is effective both in vitro and in vivo.     

In vitro transdermal iontophoretic transport of timolol maleate: effect of age and species.

Transdermal iontophoresis would be a promising method for the systemic delivery of water soluble and ionic drugs of relatively high molecular size, including peptides. In the present study, the effect of biological parameters such as age of the animal and species variation (rat, rabbit, mouse, guinea pig and human) on the transdermal iontophoretic transport was studied using timolol maleate (TM) as a model drug. The iontophoretic transport of TM across the skins obtained from the rats of different age groups was found to be similar. The results of the present study suggest that the age of the animal (Wistar rats: 1-8 months) did not appear to influence the transdermal iontophoretic transport of TM significantly. The amount of TM transported during iontophoresis (2 h) was significantly different among the different skin species. But the total amount of TM transported up to 24 h (2 h iontophoresis+22 h post-iontophoretic passive diffusion) was not significantly different among the different species studied. The present study provides further evidence that iontophoresis technique reduces the interspecies differences in the transdermal permeation of drugs, which is normally observed in passive diffusion of drugs. However, it must be noted that excised skins have been used in the present study to investigate the role of age and species variation on the iontophoretic transport of TM. The influence of these parameters under in vivo conditions might be different considering the physiological differences in different species and in the animals of different age groups.     

Preliminary studies on the use of carbon substrate utilization patterns for identification of Brucella species.

A commercial system (Biolog, Hayward, CA) that uses reduction of an indicator dye to determine the oxidation of 95 different carbon substrates contained in a defined minimal medium was tested with 35 isolates of Brucella spp. to determine if the system could be used in place of respirometric methods to identify species. Of 95 substrates contained in this system, three were oxidized by all the Brucella strains tested, 48 were oxidized by none of the strains tested, and 44 were oxidized differentially. Brucella melitensis, B. abortus, and B. suis could be distinguished from each other on the basis of their oxidation reactions in seven in these substrates; epidemiologically related strains could not be unambiguously differentiated. This carbon substrate utilization method may prove to be a useful alternative to respirometry as a means to identify strains of Brucella spp. to species level, provided that personnel are protected from exposure to this highly infectious agent.     

Methadone implants for methadone maintenance treatment. In vitro and in vivo animal studies.

Methadone implant formulations elaborated with polylactide-co-glycolide (PLGA) and polylactic acid (PLA) for 1 week and 1 month release duration, respectively, were evaluated in vitro and in vivo. One-week implants prepared with methadone clorhydrate, methadone clorhydrate/methadone base blend or methadone base were tested in vitro. Results showed that the methadone release rate decreased as the methadone base increased. The best release profile was achieve when the methadone base implants, made by compression of a 50:50 PLGA (12 kDa) and methadone base mix, were coated with PLA (30 kDa). For 1-month implants, the methadone base load was increased to 65% and PLA of 30 kDa was used as a matrix component. In this case the implants were coated with the same polymer. Deconvolution methods could not be used for in vivo release estimation because an increase in methadone clearance was observed with methadone clorhydrate solution multiple-dose treatment. Therefore the amount of drug remaining within the implants was evaluated and the deconvolution was only used to establish the release profile range. The upper limit was estimated applying the absorption-disposition function obtained after multiple-dose administrations while the lower curve was estimated using the single-dose function. Methadone serum levels were maintained around 200 ng/ml during 1 week and approximately 5 weeks with the optimised implants. In vivo-in vitro correlations were always very good with slopes near 1.     

In vitro and in vivo evaluation of the antimicrobial activity of azithromycin againstLegionella species

The antimicrobial activity of azithromycin (AZM) was evaluated againstLegionella species using an experimental model of legionellosis. The minimum inhibitory concentration₉₀s (MIC₉₀s) of AZM against 35 standard strains (0.25 mg/L) and 22 Japanese clinical isolates ofLegionella pneumophila (0.063 mg/L) were lower than those of erythromycin (EM) and almost equal to those of clarithromycin and roxythromycin. Using¹⁴C-labeled antibiotic, AZM and EM were observed concentrated inside human polymorphonuclear leukocytes (PMN) with intracellular/extracellular concentration ratios of AZM and EM at 120 minutes after dosing of 27.3 and 22.2, respectively. AZM inhibited the growth ofL. pneumophila (80-045 strain) from cultured guinea pig alveolar macrophages, even when the extracellular AZM was washed out on day 2 of culture. However, the addition of identical concentrations of EM failed to produce a similar inhibition. Pharmacokinetic studies of AZM tissue distribution in guinea pigs infected withL. pneumophila 80-045 revealed high drug concentrations in the lung and liver and a longer half-life in these organs compared with those in plasma. Treatment of guinea pigs with experimentally-induced legionellosis with oral AZM (10 mg/kg/day for 2 days) was more effective than EM treatment (10 mg/kg/day for 4 days) or a placebo, and resulted in a significant improvement in the survival rate. Our results suggest that AZM is a promising new drug for the treatment of legionellosis using a short-term dosing regimen.     

Leukotrap,a device for white cell poor platelets quality control studies In vitro and In vivo

Most febrile transfusion reactions are due to leucoagglutinins. Cutter's Leukotrap platelet pooling bag has a distal conic pouch for depleting the platelets of white blood cells by centrifugation. We tested 33 Leukotraps each containing six platelet units in vitro and 32 in vivo. The mean in vitro platelet count was 3.7 ± 0.5 × 10¹¹ platelets before, and 3.0 ± 0.5 × 10¹¹ after spinning, representing a platelet recovery of 80.2 ± 9.6% Mean white blood cells were 3.8 ± 0.6 × 10⁸ before, and 0.6 ± 0.1 × 10⁸ after centrifugation, this constituting a white cell removal of 83.5 ± 7.7%. pH ranged from 7.37 for 24-h platelets to 7.19 for 96-h platelets. 24-h after platelet pooling, all Leukotraps were sterile. Platelet aggregation with physiologic agents showed little change compared to individual platelet units. Glucose ranged between 418 and 336 mg/dL, pCO₂ between 27.8 and 19.1 mmHg, but pO₂ dropped drastically from 74.8 mmHg to 11.6 mmHg. Hypotonic osmotic recovery was satisfactory. In vivo studies were carried out with pooled, leucocyte-poor platelets which were transfused to six bone marrow transplant patients with no splenomegaly or septicemia at the outset. These patients had all demonstrated febrile transfusion reactions to standard donor units. The mean platelet increment was 16.8 × 10⁹/L. A single febrile transfusion reaction witnessed in one patient, was accompanied by an adequate platelet response. Hence Leukotrap is a useful clinical tool for reducing febrile transfusion reactions related to white blood cells.     

In vitro and in vivo activities of synthetic trioxolanes against major human schistosome species

Schistosomiasis is a parasitic disease that remains of considerable public health significance in tropical and subtropical environments. Since the mainstay of schistosomiasis control is chemotherapy with a single drug, praziquantel, drug resistance is a concern. Here, we present new data on the antischistosomal properties of representative synthetic 1,2,4-trioxolanes (OZs). Exposure of adult Schistosoma mansoni for 24 h to a medium containing 20 mug/ml OZ209 reduced worm motor activity, induced tegumental alterations, and killed worms within 72 h. While exposure of S. mansoni to OZ78 had no apparent effect, addition of hemin reduced worm motor activity and caused tegumental damage. Administration of single 200-mg/kg of body weight oral doses of OZ78, OZ209, and OZ288 to mice harboring a juvenile S. mansoni infection resulted in worm burden reductions of 82.0 to 95.4%. In the adult infection model in mice, single 400-mg/kg doses of these compounds resulted in a maximum total worm burden reduction of 52.2%. High worm burden reductions (71.7 to 86.5%) were observed after administration of single 200-mg/kg doses of OZ78 and OZ288 to hamsters infected with either juvenile or adult S. mansoni. A single 200-mg/kg dose of OZ78 to hamsters infected with adult Schistosoma japonicum resulted in total and female worm burden reductions of 94.2 to 100%. Our results, along with the low toxicity, metabolic stability, and good pharmacokinetic properties of the OZs, indicate the potential for the development of novel broad-spectrum antischistosomal OZ drug candidates.     

In vivo and in vitro studies of urinary concentrating ability in potassium-depleted rabbits.

The factors responsible for the urinary concentrating defect associated with the potassium-depleted (KD) state are uncertain. The present studies were designed to, first, determine whether a urinary concentrating defect exists in potassium-depleted rabbits and, second, to use the technique of in vitro perfusion to evaluate directly the antidiuretic hormone (ADH) responsiveness of cortical collecting tubules (CCT) in this setting. Feeding female New Zealand White rabbits a potassium-deficient diet for 2 wk caused a significant fall in plasma potassium levels in both the ad-libitum and controlled water intake groups (P less than 0.001). Muscle potassium content after 2 wk of potassium restriction fell from 45.6 +/- 0.9 to 29.0 +/- 1.2 meq/100 g fat-free dry solids (P less than 0.001). Renal papillary sodium content fell significantly from a control value of 234.6 +/- 8.0 to 182.46 +/- 10.0 meq/kg H2O after 2 wk of potassium restriction. Maximal urinary osmolality measured after 12 h of dehydration and 1.25 U pitressin IM was significantly decreased in rabbits after 2 wk of potassium restriction in both the ad-libitum and controlled water intake groups (P less than 0.001). The relationship between plasma potassium concentration and maximum urinary osmolality was significantly correlated in both the ad-libitum and controlled water intake groups, r = 0.73 and 0.68 (P less than 0.001), respectively. In addition, refeeding KD rabbits with normal chow for 1 wk resulted in normalization of both plasma potassium levels and urinary concentrating ability. CCT from control and KD rabbits were perfused in vitro at 25 degrees C. The hydraulic conductivity coefficient, Lp, was significantly reduced at all doses of ADH tested in tubules from KD rabbits when compared with control tubules. In addition, the maximal hydraulic conductivity in tubules from KD rabbits when tested with 200 microU/ml ADH at 37.5 degrees C was only 23% of control values (P less than 0.05). Furthermore, this reduced ADH responsiveness persisted when the bath potassium was elevated from 5 to 20 mM. The reflection coefficient for NaCl when compared with raffinose was 0.91 in tubules from KD animals. Thus, these data suggest that the ADH-resistant urinary concentrating defect associated with potassium depletion is due, at least in part, to a diminished responsiveness of the CCT to ADH. Therefore, further studies were designed to investigate the cellular steps involved in this abnormal response. There was no difference in the 8-para-chlorophenylthio cyclic AMP induced hydroosmotic response between CCT from KD and control rabbits. Since the cAMP-induced hydroosmotic response was similar between KD and control CCT, experiments were performed to evaluate the contribution of phosphodiesterase (PDIE) activity by using the potent PDIE inhibitor isobutylmethylxanthine (10(-4) and 10(-3)M) in the presence of ADH (200 U/ml). Although Lp was increased by PDIE inhibition in CCT from both control and KD animals, the overall hydroosmotic response in CCT from KD rabbits was still significantly reduced when compared with controls. The final experiments used forskolin to evaluate further the adenylate cyclase complex. The resulting hydroosmotic response in CCT from KD rabbits was almost identical to that obtained in controls. In conclusion, these data suggest that the decreased responsiveness of CCT from KD rabbits to ADH involves a step at or proximal to the stimulation of the catalytic subunit of adenylate cyclase, and that PDIE activity makes no contribution to this abnormal hydroosmotic response.     

Microbubble formation: In vitro and in vivo observation

Injection of liquid through a catheter into the circulation is known to produce clouds of signals detected by sonography. Blood forced through a stenotic conduit produced sonographic clouding, and bubbles of 10–100 μm were observed by light microscopy. The microbubbles persisted up to three and a half minutes. Microbubbles were observed in the microcirculation of the rat by placing the catheter tip into the descending aorta of 15 animals, viewing the mesentery at 400X magnification, and recording the results on videotape. Following injection of the rats' own blood, numerous microbubbles lodged promptly at the arteriolar level and obstructed blood flow for up to 200 sec before shrinking sufficiently to pass downstream and allow restitution of flow.     

Peri-implant care with the CO2 laser: In vitro and in vivo results

Background: Numerous applications for dental lasers have been proposed for both clinical use and experimental purposes. A new indication might be the sterilization of exposed implant surfaces in order to rehabilitate ailing implants. The purposes of this study were to assess CO₂ laser parameters for the decontamination process in vitro and to evaluate the method in vivo.Methods: In vitro, temperature changes at the bone–titanium implant interface were recorded during use of a CO₂ laser-scanning system (Swiftlase^®) and the effects of laser irradiation on titanium implants were examined. In vivo, in 6 beagle dogs, a total of 60 implants and bony defects were treated either conventionally by air-powder-abrasive or by laser irradiation or in combination to evaluate if reosseointegration can occur. In 16 patients (41 ailing implants), the reliability of the CO₂ laser-assisted vs. conventional decontamination was tested.Results: Depending on the parameters chosen, melting and other surface alterations could be seen in vitro. In continuous wave mode, mean power output of 2.5 W for a maximum of 10 s is suitable for the decontamination process. In the beagle dog model, histologic examination revealed new direct bone-to-implant contact following laser-assisted therapy. The clinical study showed 4 months after therapy that laser-decontaminated implants and soft tissue resection resulted in statistically significant better radiographic parameters than conventional decontamination plus soft tissue resection.Conclusions: From these results it was concluded that treatment of peri-implantitis can be optimized using CO₂ laser-assisted implant decontamination. Nevertheless, further studies are required in this field.

Zusammenfassung

Einleitung: In den letzten Jahren wurde eine zunehmende Zahl von Indikationen für den Einsatz von Dentallasern genannt. Eine neue Anwendung könnte in der Dekontamination freiliegender Implantatoberflächen bestehen. Ziel dieser Untersuchungen war, geeignete Laserparameter in vitro und in vivo zu identifizieren und die Methode am Patienten zu überprüfen.Material und Methode: In vitro wurden die Temperaturanstiege am Titan-Knochen-Interface während CO₂ Laser-Bestrahlung unter Verwendung eines Scanners gemessen und die Auswirkungen auf die Implantatmorphologie untersucht. In einer tierexperimentellen Studie wurden insgesamt 60 periimplantäre Defekte konventionell, durch Laserbestrahlung bzw. in Kombination therapiert und histologisch untersucht, inwieweit knöcherne Reappositionen möglich sind. Ziel einer klinischen Studie an 16 Patienten bzw. 41 Implantaten war es, die Laser-gestützte Dekontamination im Vergleich zum konventionellen Vorgehen zu evaluieren.Ergebnisse: Abhängig von den Parametern waren Aufschmelzungen der Implantatoberfläche zu erkennen. Im cw-Betrieb waren dagegen mittlere Leistungen bis zu 2,5 W für bis zu 10 s Bestrahlungszeit applizierbar, ohne die kritische Temperatur am Interface zu überschreiten. Im Tiermodell zeigten sich nach 4 Monaten knöcherne Reappositionen an vormals kontaminierten Implantatoberflächen. In einer klinischen 3-Jahres-Studie waren nach Lasertherapie bessere röntgenologische Parameter nachweisbar als nach konventioneller Dekontamination.Konklusion: Aufgrund dieser Ergebnisse wurde die Schlussfolgerung gezogen, dass die CO₂-Laser-assistierte Implantatdekontamination die Therapie periimplantärer Entzündungen optimieren kann. Weitere Studien sind erforderlich, um ein gesichertes Behandlungsprotokoll für die Periimplantitistherapie erstellen zu können.     

Evaluation of platelets prepared by apheresis and stored for 5 days. In vitro and in vivo studies

To evaluate the effect of storage on apheresis platelets collected with a closed-system blood cell separator, an in vitro investigation was performed, with measurements of pH, lactate, ATP, the ratio of ATP to the total adenine nucleotide content, and adenylate kinase. Unmodified apheresis platelets and apheresis platelets with plasma added were compared with conventional platelets stored in PL-1240 or PL-732 plastic containers. During 6 days of storage, there were similar changes in all variables with one exception: the extracellular activity of adenylate kinase was lower in apheresis platelets with plasma than in the other three groups (p less than 0.01). In vivo studies were carried out with 111Indium-labeled autologous platelets in eight volunteers. Apheresis platelets with 100 mL of plasma added were stored in two 1000-mL containers (PL-732) at 22 degrees C during agitation. Platelets from one of the containers were labeled with 111Indium and transfused into the volunteer within 24 hours. Platelets from the other container were labeled after 5 days of storage and transfused into the same donor. There were no significant differences between apheresis platelets stored for 1 day and those stored for 5 days: the mean percentage of recovery was 58.4 and 57.6 percent, t1/2 was 69 and 67 hours, and the survival time was 5.5 and 5.6 days, respectively.     

Cardiac muscarinic receptors decrease with age. In vitro and in vivo studies.

The M1 muscarinic receptor antagonist pirenzepine in low doses decreases resting heart rate; this effect declines with age (Poller, U., G. Nedelka, J. Radke, K. Pönicke, and O.-E. Brodde. 1997. J. Am. Coll. Cardiol. 29:187-193). To study possible mechanisms underlying this effect, we assessed (a) in six young (26 yr old) and six older volunteers (61 yr old), pirenzepine effects (0.32 and 0.64 mg intravenous [i.v.] bolus) on isoprenaline-induced heart rate increases; (b) in five heart transplant recipients, pirenzepine effects (0.05-10 mg i.v. bolus) on resting heart rate in the recipient's native and transplanted sinus nodes; and (c) in right atria from 39 patients of different ages (5 d-76 yr) undergoing open heart surgery, M2 muscarinic receptor density (by [3H]N-methyl-scopolamine binding) and adenylyl cyclase activity. (a) Pirenzepine at both doses decreased heart rate in young volunteers significantly more than in older volunteers; (b) pirenzepine (< 1 mg) decreased resting heart rate in the recipient's native but not transplanted sinus node; and (c) M2 receptor density and carbachol-induced inhibition of forskolin-stimulated adenylyl cyclase activity decreased significantly with the age of the patients. We conclude that pirenzepine decreases heart rate via inhibition of presynaptic M1 autoreceptors, thereby releasing endogenous acetylcholine, and that the heart rate-decreasing effect of acetylcholine declines with age because right atrial M2 receptor density and function decrease.     

In vitro and in vivo studies of the effect of artemether on Schistosoma mansoni.

To determine whether artemether, a derivative of the antimalarial agent qinghaosu, is therapeutically active against Schistosoma mansoni, we determined the in vitro, in vivo, and histopathologic effects of the drug on S. mansoni worms. In vitro, toxic effects of artemether on S. mansoni were not seen at concentrations of less than 100 micrograms/ml. However, in vivo, 30 and 50% reductions in the lengths of male and female worms, respectively, were observed 14 days after treatment. By 56 days worm dimensions had returned to control values. Similar reversible effects on male testes and female ovaries were seen. In vivo, a single oral dose of artemether (300 mg/kg) induced a shift of worms towards the liver within 8 h after treatment. By 3 and 14 days after treatment, 99 and 76%, respectively, of worms were still in the liver. In vivo, the therapeutic effect of artemether on adult S. mansoni treated on day 56 after infection was modest. Doses as high as 1,200 mg (200 mg/kg per day, six doses) resulted in a worm reduction rate of only 39%. However, in infected mice treated on day 14 or 21 after infection, worm reduction rates of 83 to 98% were obtained. Thus, artemether exhibited modest in vitro and in vivo activities against adult S. mansoni but was twofold more active against 2- to 3-week-old liver-stage parasites.