Changes in the excretion of endogenous glycine conjugate as a possible artifact in toxicological experiments

Changes in the urinary excretion of hippuric acid (HIA) and phenaceturic acid (PUA) as well as their metabolic precursors, i.e. benzoic (BA) and phenylacetic acid (PAA), in rats housed in glass metabolic cages for 4 days were monitored using gas-liquid chromatography. The amount of HIA excreted was 128±63 mol/kg for female and 79±43 mol/kg for male rats in the first 24 h and decreased to 11±7 mol/kg (p< 0.01) for female and 3.2±2.4 mol/kg (p< 0.001) for male rats on the 2nd day. These values remained nearly at the same level until the end of the experiment. The amount of PUA decreased from 48±12 mol/kg on the 1st day to 22±9 mol/kg (p< 0.05) on the 2nd day by male rats, whereas by the females the decrease from 30±9 mol/kg to 21±8 mol/kg was not significant. The decrease in the excretion of glycine conjugates was compensated by a parallel increase in the level of unconjugated BA and PAA.     

Cocaine inhibits cromakalim-activated K<Superscript>+</Superscript> currents in follicle-enclosed<Emphasis Type="Italic"> Xenopus</Emphasis> oocytes

The effect of cocaine on K⁺ currents activated by the K_ATP channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that cocaine in the concentration range of 3–500 M reversibly inhibits cromakalim-induced K⁺ currents. The IC₅₀ value for cocaine was 96 M. Inhibition of the cromakalim-activated K⁺ current by cocaine was noncompetitive and voltage independent. Pretreatment with the Ca²⁺ chelator BAPTA did not modify the cocaine-induced inhibition of cromakalim-induced K⁺ currents, suggesting that Ca²⁺-activated second messenger pathways are not involved in the actions of cocaine. Outward K⁺ currents activated by the application of 8-Br-cAMP or forskolin were also inhibited by cocaine. The EC₅₀ and slope values for the activation of K⁺ currents by cromakalim were 184±19 M and 1.14 in the absence of cocaine as compared to 191±23 M and 1.03 in the presence of cocaine (300 M). Cocaine also blocked K⁺ currents mediated through C-terminally deleted form of Kir6.2 (KirC26) in the absence of sulfonylurea receptor with an IC₅₀ value of 87 M, suggesting that cocaine interacts directly with the channel forming Kir6.2 subunit. Radioligand binding studies indicated that cocaine (100 M) did not affect the binding characteristics of the K_ATP ligand, [³H]glibenclamide. These results demonstrate that cromakalim-activated K⁺ currents in follicular cells of Xenopus oocytes are modulated by cocaine.     

Disposition of orally administered di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate in the rat

The dispositon of di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) was studied in the rat. Three hours after a single oral dose of DEHP (2.8 g/kg), plasma concentrations of 8.8±1.7 g/ml DEHP and 63.2±8.7 g/ml MEHP were reached. MEHP levels declined with a half-life of 5.2±0.5 h. The ratio of the area under the plasma concentration-time curve of MEHP to that of DEHP was 16.1±6.1. When ¹⁴CDEHP was administered, 19.3±3.3% of the radioactivity was excreted in the urine within 72 h, the rest being excreted in the faeces. The urinary excretion rate of total radioactivity declined with a half-life of 7.9±0.5 h. Single administration of MEHP (0.4 g/kg) resulted in plasma concentrations of 84.1±14.9 g/ml 3 h after dosing; the half-life of MEHP was 5.5±1.1 h. Multiple dosing with DEHP (2.8 g/kg/day) for 7 consecutive days produced no accumulation of DEHP or MEHP in plasma.     

Lead and cadmium in breast milk higher levels in urban vs rural mothers during the first 3 months of lactation

Breast milk from 10 women each from the city of Hamburg and from a rural area was analyzed by atomic absorption spectrometry for contamination with lead and cadmium. Samples were examined at regular intervals for 3 months after birth. On day 5 a diurnal profile was analyzed; on the other days milk was taken before and after the morning feed.Daily permissable intake (DPI) for lead is 5 g/kg/day for children; the DPI for cadmium has as yet been determined only for adults as 400–500 g/week, equivalent to about 1 g/kg/day (WHO 1972). For breast milk as the main source of nutrition in infants, this study shows values of 9.1±2.5 (SD) g/l for lead in the rural population, with a tendency to decrease towards the end of lactation. Urban mothers had 13.3±5.5 (SD) g/l, with a tendency to increase. This difference was significant only on day 45. Mean cadmium content in rural mothers was 17.3±4.9 g/l, with much higher values in the colostrum and a decrease after 15 days. Urban mothers had 24.6±7.3 g/l, again with high colostrum values and a subsequent decrease. These latter values are not significantly different.Calculated daily intake according to these values is presented, based on 840 ml breast milk for a 5.5 kg infant per day. Rural infants ingested 0.9–1.3 g/kg/day of lead, and in the city 1.5–2.3 g/kg/day. Cadmium intake in rural infants amounted from 1.2–1.8 g/kg/day; in Hamburg it was 1.6–2.2 g/kg/day. Thus the daily ingestion of lead was just below the DPI, cadmium ingestion was higher than the DPI for adults. The rural population had lower values in breast milk for both heavy metals than the urban population, although not statistically significant. Compared to earlier reports there was a slight increase in lead concentration and a more significant increase for cadmium. This study shows that the increase in cadmium may have taken place during very recent years, possibly due to the increase in the pH of soil. Therefore, it is suggested that both levels be monitored in a continuous program to prevent any nutritional hazard.     

Actions of cisplatin on the electrophysiological properties of cultured dorsal root ganglion neurones from neonatal rats

In this study we have employed the whole cell patch clamp technique to investigate the effects of an anticancer drug cisplatin on basic electrophysiological properties of cultured dorsal root ganglion neurones from neonatal rats. The results show that within the clinical concentration range, cisplatin (0.1 to 10 M) caused a decrease in input conductance, and complex changes in resting membrane potential in these cultured sensory neurones. The dominant effects of cisplatin on input conductance may be due to inhibition of leak conductances. Transplatin (5 M) was significantly less effective than cisplatin at reducing input conductance which suggests a degree of stereo selectivity.Cisplatin (1 to 5 M) transiently increased excitability of the cultured neurones as reflected by a reduction in the threshold for activation of action potentials by 8 mV. The rise time, peak amplitude and duration of action potentials were not changed by acute application of 5 M cisplatin.Long term treatment of neurones with cisplatin (5 M), for up to 1 week reduced the viability of the cultures, and attenuated neurone excitability, although input conductance of the cells was significantly increased to 322±49 M (n = 9) compared with controls of 210±20 M (n = 30; P<0.05). Acute and chronic treatment of cultured neurones with cisplatin therefore produced contrasting actions. Correspondence to: R. H. Scott at the above address     

Stereoselective analysis of the disposition of tosufloxacin enantiomers in man

Summary The pharmacokinetics of tosufloxacin enantiomers after oral administration of racemic tosufloxacin were examined in healthy volunteers. Only small differences were observed in time to peak concentration (2.6±0.3 [mean ± SEM] h for (+)-tosufloxacin vs 2.4±0.2 h for (–)-tosufloxacin), elimination half-life (3.61±0.24 h vs 3.49±0.23 h), and area under the curve (2.78±0.19 h·g/ml vs 2.87±0.19 h·g/ml); however, peak concentration (0.40±0.03 g/ml vs 0.44±0.03 g/ml), renal clearance (226±10 ml/min vs 202±10 ml/min), and urinary recovery (35.4±2.2% vs 32.4±1.9%) differed significantly between enantiomers.     

Influence of N-allyl-normetazocine on acetylcholine release from brain slices: involvement of muscarinic receptors

Summary The effects of (±)N-allyl-normetazocine on the release of acetylcholine from different areas of guinea-pig and rat brain were investigated. 1. The drug did not modify the electrically (2 Hz) evoked tritium efflux from guinea-pig cerebral cortex, thalamus and caudate nucleus slices, preloaded with ³H-choline 0.1 mol/l and superfused with Krebs solution containing hemicholinium-3 10 mol/l. 2. (±)N-allyl-normetazocine 10 mol/l. enhanced the evoked ³H efflux from guinea-pig brain slices superfused with Krebs solution containing physostigmine 30 mol/l or oxotremorine 0.3 -1 gmol/l; the effect was naloxone-insensitive and was abolished by atropine 0.15 mol/l, but not by pirenzepine 1 mol/l. 3. (±)N-allyl-normetazocine 5 mol/l enhanced the electrically evoked release of endogenous acetylcholine as well, in a naloxone-insensitive way. 4. Both (±) and (+)N-allyl-normetazocine were without effect on ³H efflux from rat caudate nucleus slices electrically stimulated at 0.2 Hz frequency, after preloading with ³H-choline and during superfusion with hemicholinium-3. 5. The results are discussed in view of the antimuscarinic properties of the drug. Send offprint requests to A. Siniscalchi     

In Vitro Cutaneous Disposition of a Topical Diclofenac Lotion in Human Skin: Effect of a Multi-Dose Regimen

Purpose. This study determines comparative bioavailability of diclofenac sodium lotion compared to an aqueous solution after topical application to viable human skin in vitro. In addition, the difference between a single dose and multiple doses (8 times) was also determined. Methods. An in vitro flow-through diffusion cell system was employed, using radiolabelled diclofenac sodium. Results. Multiple doses of lotion (2 l/cm² and 5 l/cm²) delivered a total of 40.1 ± 17.6 g and 85.6 ± 41.4 g diclofenac, respectively, at 48 h, compared to only 9.4 ± 2.9 g and 35.7 ± 19.0 g absorbed after topical application of diclofenac as an aqueous solution (P < 0.05). A single dose study showed no statistical difference between diclofenac delivered in lotion or an aqueous solution. Over 48 h the total absorption from lotion was 10.2 ± 6.7 g and 26.2 ± 17.6 g (2 l/cm² and 5 l/cm², respectively), compared to 8.3 ± 1.5 g and 12.5 ± 5.7 g from an aqueous solution. Both single doses of lotion and aqueous diclofenac showed decreased diclofenac absorption into the receptor fluid between 12 and 24 h. However, when applied multiple times, absorption from lotion was continually increasing up to 48 h. The total dose accountability ranged from 76.8 ± 8.2% to 110.6 ± 15.1% of the applied dose. Conclusions. Diclofenac lotion exhibited enhanced diclofenac percutaneous absorption rate through human skin (mass, flux and partition coefficient) when applied a multiple number of times and this enhanced absorption was maintained over 48 h. This suggests that a constituent of the lotion (DMSO) will enhance human skin absorption of diclofenac when used in a multi-dose regimen, but not after a single dose.     

Carrier Mechanisms Involved in the Transepithelial Transport of bis(POM)-PMEA and Its Metabolites Across Caco-2 Monolayers

Purpose. To investigate the role of carrier mechanisms in: [1] the polarized transport of the bis(pivaloyloxymethyl)- [bis(POM)-] ester prodrug of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine [PMEA] and [2] the directional secretion of its metabolites. Methods. Caco-2 monolayers were used to study the modulating effect of carriers on the transport of bis(POM)-PMEA and the efflux of intracellularly formed metabolites mono(POM)-PMEA and PMEA from the cells. The interaction of bis(POM)-PMEA and its metabolites with the efflux mechanisms present in Caco-2 monolayers was investigated by testing the effect of various concentrations of verapamil (30, 100, 300 M) or indomethacin (10-500 M) on transport and efflux. Results. Polarity in transport of bis(POM)-PMEA (50 M) across Caco-2 monolayers was noted: transport of total PMEA [=bis(POM)-PMEA, mono(POM)-PMEA and PMEA] was significantly higher in basolateral (BL) to apical (AP) direction (14.5 ± 0.4%) than transport in the opposite (AP to BL) direction (1.7 ± 0.2%). This difference was reduced in a concentration dependent way when verapamil (0–100 M) was included in both AP and BL incubation media. After loading the cells with bis(POM)-PMEA (100 M) for 1 hr, studies on efflux of PMEA and mono(POM)-PMEA from the Caco-2 monolayers over a 3 hr period, revealed that both metabolites were preferentially secreted towards the AP compartment. Efflux of PMEA towards AP and BL compartments amounted to 14.6 ± 1.1 % and 5.3 ± 0.4%, respectively, of the initial intracellular amount of total PMEA, while efflux of mono(POM)-PMEA towards AP and BL compartments was limited to 2.3 ± 0.1 % and 0.5 ± 0.1 %, respectively. When 10 M indomethacin was included in the AP incubation medium, efflux of PMEA was decreased to 7.8 ± 0.3% and 3.3 ± 0.3% towards the AP and BL compartments, respectively. The decrease in efflux by indomethacin was concentration-dependent up to 100 M. Transepithelial transport of total PMEA was also reduced in the presence of 30 M indomethacin, as reflected in smaller concentrations of PMEA and mono(POM)-PMEA in the acceptor compartment, irrespective of the transport direction. Conclusions. The data obtained in this study suggest that bis(POM)-PMEA is substrate for a P-glycoprotein-like carrier mechanism in Caco-2 monolayers, while its metabolites mono(POM)-PMEA and PMEA are transported by a non-P-glycoprotein efflux protein.     

Effects of (+)- and (±)-sotalol on repolarizing outward currents and pacemaker current in sheep cardiac Purkinje fibres

Summary This study was aimed to differentiate the action of (+)- and (±)-sotalol (10–1000 mol/l) on membrane currents which are active during the repolarization of cardiac action potentials Effects where studied in shortened sheep cardiac Purkinje fibres with the two-microelectrode voltage-clamp technique Action potentials were activated at a frequency of 0.25 Hz and membrane currents at 0.03 Hz or 0.05 Hz in most experiments.Out of the currents investigated the transient outward current (i_to) reacted most sensitively to (+)- and (±)-sotalol. I_to-amplitude was decreased on the average to 77% of reference at 10 mol/l and to 53% at 1000 mol/l (+)- or (±)-sotalol. The maximally available ito-current was decreased but the voltage-dependent control of inactivation was left nearly unchanged. The initial inwardly rectifying current (i_Ki), which propels the last repolarization phase of the action potential and controls resting potential to a large extent was reduced on the average to 93% of reference at 10 mol/l and to 62% at 1000 mol/l (+)- or (±)-sotalol. Time-dependent (delayed) outward current (i_K) was on the average not affected by (+)- or (±)-sotalol up to 100 mol/l and was decreased to 84% of reference current under the influence of 1000 mol/l. An initial outward current, which is activated at positive membrane potentials (i_inst) was not clearly affected by (+)- or (±)-sotalol at concentrations up to 1000 mol/l Pacemaker current (i_f) was not influenced by the drugs up to 100 mol/l. Only at 1000 mol/l was the amount of available i_f-current decreased to 79% of reference. (The potential-dependent control of activation was not affected) Time constants of time-dependent currents i_to, i_K and i_f did not change in concentrations up to 1000 mol/l of the drug.Action potential duration increased at (+)- or (±)-sotalol concentrations 10 mol/l and maximal prolongation was achieved at concentrations of 100–300 mol/l Resting potential remained nearly unchanged at these concentrations, but the membranes depolarized at 1000 mol/l. According to our data action potential prolongation in sheep Purkinje fibres under the influence of (+)- and (±)-sotalol correlates to the drug-induced block to i_to-current and inwardly rectifying i_K1-current.Supported by the Deutsche Forschungsgemeinschaft SFB 242, C 1 Send offprint requests to U. Borchard at the above address     

Nicorandil attenuates the mitochondrial Ca<Superscript>2+</Superscript> overload with accompanying depolarization of the mitochondrial membrane in the heart

The anti-anginal drug nicorandil has been demonstrated to protect the myocardium against ischemic injury in both experimental and clinical studies. Although nicorandil seems to protect the myocardium via activation of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels, the mechanisms underlying its cardioprotection have remained elusive. We therefore examined whether nicorandil depolarizes the mitochondrial membrane and attenuates the mitochondrial Ca²⁺ overload. With the use of a Nipkow confocal system, the mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) and the mitochondrial membrane potential (_m) in rat ventricular myocytes were measured by loading cells with rhod-2 and JC-1 respectively. The number of cell hypercontractures resulting from mitochondrial Ca²⁺ overload was counted. Exposing cells to ouabain (1 mM) evoked mitochondrial Ca²⁺ overload and increased the intensity of rhod-2 fluorescence to 180±15% of baseline (p<0.001). Nicorandil (100 M) significantly attenuated the ouabain-induced mitochondrial Ca²⁺ overload (129±4% of baseline; p<0.001 vs. ouabain). Nicorandil decreased the _m during application of ouabain, thereby reducing the intensity of JC-1 fluorescence to 89±2% of baseline (p<0.05). Exposure of myocytes to ouabain eventually resulted in cell hypercontracture (51±2%). This ouabain-induced cell hypercontracture was blunted by application of nicorandil (37±2%, p<0.05 vs. ouabain). Moreover, these effects of nicorandil were abolished by 5-hydroxydecanoate (500 M), a putative mitoK_ATP channel blocker, and by glibenclamide (10 M), a nonselective K_ATP channel blocker. Our results suggest that nicorandil attenuates the matrix Ca²⁺ overload with accompanying depolarization of the mitochondrial membrane. Such effect might potentially be attributed to the mechanism of cardioprotection afforded by nicorandil.     

Phase II trial of 9-aminocamptothecin (NSC 603071) administered as a 120-hour continuous infusion weekly for three weeks in metastatic colorectal carcinoma

9-Aminocamptothecin (9-AC) is a camptothecin derivative with broad antitumor activity in preclinical studies. Prior investigations suggested that prolonged maintenance of 9-AC lactone plasma concentrations above 10 nmol/l and frequent administration of the drug are important determinants of antitumor activity. Our phase II study, therefore, examined a 5-day continuous infusion of 9-AC weekly for 3 weeks in patients with advanced colorectal cancer. Eighteen patients previously untreated for metastatic disease received 480 g/m²/day of 9-AC. No responses were observed in 17 evaluable patients. Severe toxicities included granulocytopenia, nausea, vomiting and diarrhea. The median absolute granulocyte count (AGC) nadir was 2,300/l (range 0–9,000/l) and occurred on day 10. Eight patients received an escalated dose of 600 g/m²/day. The median AGC nadir at the escalated dose was 1,500/l (range: 300–2,700/l) and occurred on day 22. The median number of courses given was 2 (range: 1–8); and the median time to disease progression was 8 weeks (range: 1–40 weeks). 9-AC administered by this schedule lacked antitumor activity in patients with advanced colorectal carcinomas.     

Mechanisms of action of plant sterols on inhibition of cholesterol absorption

Summary The effects of two different plant sterols on intestinal cholesterol absorption were compared in normal volunteers by an intestinal perfusion study during a control period followed by high dose infusion of sitosterol or sitostanol (3.6 mol/min), to which subjects were allocated in a randomized manner. Cholesterol absorption during the control period was similar in the two groups, averaging 0.88 ± 0.48 mol/min (32 ± 11%) for group I (sitosterol) and 0.68 ± 0.33 mol/min (29 ± 9%) for group II (sitostanol). The infusion of a high dose of sitosterol resulted in a significant reduction of cholesterol absorption to 0.47 mol/min (16%). Following the same dose of sitostanol, cholesterol absorption diminished significantly to 0.15 ± 0.11 mol/min (5.1 ± 2.9%). Overall cholesterol absorption declined during sitosterol infusion by almost 50%, whereas sitostanol infusion caused a reduction of cholesterol absorption by almost 85%. These findings of a more effective inhibition of cholesterol absorption by sitostanol might confirm the observation recorded by others that an increase in hydrophobicity of a plant sterol results in a higher affinity but lower capacity to mixed micells. This may cause an effective displacement of cholesterol from micellar binding and therefore diminished cholesterol absorption.     

The depolarizing action of 5-hydroxytryptamine on rabbit isolated preganglionic cervical sympathetic nerves

Summary This study describes a depolarizing action of 5-hydroxytryptamine (5-HT) on rabbit isolated preganglionic cervical sympathetic nerves using an extracellular recording technique. From cumulative concentration-response curves for 5-HT (1 mol/1-1 mmol/1), the mean maximal depolarization was shown to be 277 ± 32 V and EC₅₀ was 9.4 mol/l(6.5–13.6 mol/l, geometric mean, 95% confidence limits, n = 42). The responses to 5-HT displayed marked tachyphylaxis. When cumulative concentration-response curves to 5-HT and 2-methyl-5-HT were determined in the same preparations (n = 4), the mean maximal response to 5-HT was 519 ± 167 V, EC₅₀ 32.2 mol/l (8.8–118 mol/l) and the mean maximal response to 2-methyl-5-HT was 317 ± 63 V, EC₅₀ 35.1 mol/l (12.9–95.5 mol/l, geometric means, 95 % confidence limits). The action of selective 5-HT antagonists was tested on repeated cumulative concentration-response curves to 5-HT. Neither methiothepin (0.1–1 mol/l, _n = 3) nor ketanserin (0.1–1 mol/l, n = 3) had an action on 5-HT responses. The selective 5-HT₃ antagonists MDL 72222, ICS 205-930 and SDZ 206–830 were all potent antagonists of the 5-HT depolarizations. The action of these antagonists was quantified by determining the apparent pA₂ from the dose ratios and a Schild plot. For MDL 72222 (1 nmol/1-0.1 mol/l), the apparent pA₂ was 9.1 ± 0.1 (n = 12), Schild plot: 9.2; for ICS 205–930 (0.1 nmol/l–3 nmol/1), the apparent pA₂ was 10.4 ± 0.1 (n = 11), Schild plot 10.3, and for SDZ 206–830 (0.03 nmol/l-1 nmol/1), the apparent pA₂ was 11.2 ± 0.1 (n = 12), Schild plot 11.2. 5-HT depolarizations were unaffected by hexamethonium (0.5 mmol/1). 5-HT depolarizations were reduced by superfusion with both Na-free (42 ± 8% of controls, n = 4) and Na/Ca-free media (35 ± 7% of controls, n = 4). It is concluded that 5-HT depolarizations of rabbit preganglionic cervical sympathetic nerve are mediated by 5-HT₃ receptors. The data with selective 5-HT₃ receptor antagonists suggest that the receptor profile may be more like that for the 5-HT₃ receptor on the terminals of sympathetic nerves than that for the 5-HT₃ receptor on the soma of superior cervical ganglion cells or on vagal afferent neurones. Send offprint requests to D. I. Wallis at the above address     

Effects of dichlorvos (DDVP) inhalation on the activity of acetylcholinesterase in the bronchial tissue of rats

The experiments presented here deal with the effects of the inhalation of dichlorvos [dimethyl-(2,2 dichlorvinyl)-phosphate, DDVP] vapor on acetyl-cholinesterase (ACHE) activity in rat bronchial tissue. Exposure to DDVP concentrations of 0.8 and 1.8 g/l for 3 days reduced ACHE activity in the bronchial tissue (62.8±0.8 and 51.6±1.6% of the control), but did not elicit any changes in blood ACHE activity (101±4.5% of the control each). Higher concentrations (4.3 g/l) induced a decline in ACHE activity also in the blood (38.2±1.1% of the control). In the histochemical preparations used to demonstrate CHE activity in bronchial tissue (thiolacetic acid method), a staining of the bronchial glands and smooth muscles characteristic of the enzyme activity was strongly reduced after exposure of the animals to even the lowest dose applied (0.2 g/l). The question of whether localized inhibition of ACHE in the bronchial tissue might cause increases in airway resistance due to activation of a broncho-bronchial reflex is discussed. This efferent cholinergic mechanism has been found to be at least partly responsible for maintenance of bronchospasm and hypersecretion in chronic obstructive deseases of the respiratory system.     

Biodegradable PLGA Microspheres Loaded with Ganciclovir for Intraocular Administration. Encapsulation Technique,in Vitro Release Profiles,and Sterilization Process

Purpose. The purpose of this work was to obtain a sterilized formulation consisting of biodegradable microspheres of poly (DL-lactide-co-glycolide) (PLGA) for intraocular sustained release of ganciclovir. Methods. Microspheres were prepared using a dispersion of ganciclovir in fluorosilicone oil (FSiO) that was further dispersed in an acetone solution of PLGA [50/50 and inherent viscosity 0.41 dl/g], and emulsified in silicone oil with a surfactant. Once prepared, the formulation was exposed with an effective radiation dose of 2.5 megarads. The release rate data of ganciclovir from the sterilized and nonsterilized batches were compared using the similarity factor (f₂). Results. The dispersion of the drug in FSiO contributed to achieving a drug payload of up to 95% of the theoretical in the 300-500 m microspheres. Ten mg released ganciclovir in vitroat 1.3 g/h for the first 21 days, but decreased to 0.2 g/h from day 25 until the end of the release study (42 days). No significant differences in the amounts of encapsulated drug (=0.05) were observed between the sterilized and nonsterilized microspheres. Furthermore, dissolution profiles of formulations behaved similarly before and after gamma radiation exposure. Conclusions. The technique of microsphere preparation described resulted in high ganciclovir loading (95%) and prolonged drug release. The ganciclovir formulation behaved similarly before and after the sterilization process.     

Effect of BHT 920 on monoaminergic neurons of the rat brain: an electrophysiological in vivo and in vitro study

Summary BHT 920 was originally described as a dopamine autoreceptor agonist. In this study, the effect of this compound on the firing rate of noradrenergic locus coeruleus, serotonergic dorsal raphe and dopaminergic ventral tegmental area neurons was examined both in the anaesthetized rat and in rat brain slices. Extracellular recordings were performed in cells whose identity was determined by electrophysiological, pharmacological and histological criteria. In vivo, BHT 920 inhibited the firing of locus coeruleus neurons (ID 50: 14.5 ± 4.7 g/kg, mean ± SEM) and ventral tegmental area neurons (ID50 7 ± 3 g/kg) at very low doses. As a comparison, the ID50 of clonidine on locus coeruleus cells was 5.5 ± 0.6 g/kg and the ID50 of apomorphine on ventral tegmental area neurons was 13 ± 3 g/kg. BHT 920 also decreased the firing of dorsal raphe cells, but this effect was obtained at higher doses (ID50: 57 ± 11 g/kg).The in vitro study confirmed the results obtained in vivo. BHT 920 potently inhibited the firing of locus coeruleus cells (IC50: 71 ± 28 nM) and was less potent than clonidine (IC50: 5.3 ± 0.98 nM). The compound also inhibited the firing of ventral tegmental area neurons at very low concentrations (IC50: 21 ± 3.3 nM), being more potent than apomorphine (IC50: 56 ± 29 nM).BHT 920 only slightly decreased the firing rate of dorsal raphe neurons at 50 gM, showing that the drug has little direct effect on these cells.A pharmacological analysis performed in vitro showed that the effect of BHT 920 was specifically inhibited by the D₂ antagonist sulpiride (1 M) in the ventral tegmental area and by the alpha₂ antagonist idazoxan (1 M) in the locus coeruleus.This electrophysiological study shows that BHT 920 is a potent D₂ and alpha₂ agonist in the rat brain.Send offprint requests to Prof. A. Dresse at the above address     

The relaxant effects of cromakalim (BRL 34915) on human isolated airway smooth muscle

Summary Cromakalim (BRL 34915) is a potassium channel opener with therapeutic potential as a bronchodilator in asthma. Cromakalim (0.1–30 mol/l) inhibited the spontaneous tone of human isolated bronchi n a concentration-related manner being nearly as effective as isoprenaline or theophylline. The order of relaxant potencies (expressed as -log₁₀ IC₅₀ mol/l; mean ±SEM) was isoprenaline (7.29 ± 0.27; n = 8) > cromakalim (5.89 ± 0.12; n = 7) > theophylline (4.07 ±0.13; n = 10). In human bronchi where tone had been raised by addition of histamine (0.1 mmol/l), acetylcholine (0.1 mmol/l) or leukotriene D₄ (LTD₄, 0.1 mol/l), the relaxant effect of cromakalim was substantially reduced. Cromakalim suppressed the contraction produced by KCI (25 mmol/l) but not that produced by KCl (120 mmol/l). Tetraethylammonium (8 mmol/l) was without effect against the relaxant action of cromakalimbut procaine (0.5 – 5 mmol/l) and glibenclamide (0.3 mol/l) antagonised it. Cromakalim (10 mol/l) produced an upward displacement of concentration-effect curves forKCI (1–100 mmol/l), acetylcholine (1 nmol/l-1 mmol/) and histamine (1 nmol/l-1 mmol/l) but it did not alter the concentration-effect curve for LTD₄ (0.1 nmol/l-0.1 mol/l). When tissues were challenged in the presence of cromakalim (10 mol/l) with KCI (100 mmol/l), acetylcholine (1 mmol/l) or histamine (1 mmol/l), an enhanced contraction was observed compared to control tissues. This enhancement by cromakalim was absent when tissues were challenged with acetylcholine or histamine in either a Ca²⁺-free medium (plus EGTA 0.1 mmol/l) or in the presence of verapamil (10 mol/l). It is concluded that cromakalim is an effective relaxant of human airway smooth muscle in vitro and this activity may depend on the opening of K⁺ channels in the plasma membrane of smooth muscle cells but other actions cannot be ruled out. Correspondence to: J. Cortijo at the above address     

Testosterone reinforcement: intravenous and intracerebroventricular self-administration in male rats and hamsters

Rationale Anabolic steroids are drugs of abuse. However, the potential for addiction remains unclear. Testosterone induces conditioned place preference in rats and oral self-administration in hamsters.Objectives To determine if male rats and hamsters consume testosterone by intravenous (IV) or intracerebroventricular (ICV) self-administration.Methods With each nose-poke in the active hole during daily 4-h tests in an operant conditioning chamber, gonad-intact adult rats and hamsters received 50 g testosterone in an aqueous solution of -cyclodextrin via jugular cannula. The inactive nose-poke hole served as a control. Additional hamsters received vehicle infusions.Results Rats (n=7) expressed a significant preference for the active nose-poke hole (10.0±2.8 responses/4 h) over the inactive hole (4.7±1.2 responses/4 h). Similarly, during 16 days of testosterone self-administration IV, hamsters (n=9) averaged 11.7±2.9 responses/4 h and 6.3±1.1 responses/4 h in the active and inactive nose-poke holes, respectively. By contrast, vehicle controls (n=8) failed to develop a preference for the active nose-poke hole (6.5±0.5 and 6.4±0.3 responses/4 h). Hamsters (n=8) also self-administered 1 g testosterone ICV (active hole:39.8±6.0 nose-pokes/4 h; inactive hole: 22.6±7.1 nose-pokes/4 h). When testosterone was replaced with vehicle, nose-poking in the active hole declined from 31.1±7.6 to 11.9±3.2 responses/4 h within 6 days. Likewise, reversing active and inactive holes increased nose-poking in the previously inactive hole from 9.1±1.9 to 25.6±5.4 responses/4 h. However, reducing the testosterone dose from 1 g to 0.2 g per 1 l injection did not change nose-poking.Conclusions Compared with other drugs of abuse, testosterone reinforcement is modest. Nonetheless, these data support the hypothesis that testosterone is reinforcing.     

Pharmacokinetics and safety of ILX23-7553, a non-calcemic-vitamin D3 analogue, in a phase I study of patients with advanced malignancies

Purpose: Differentiation therapy is an alternative to chemotherapy with potentially less toxicity, improved quality of life, and survival. We conducted a phase I trial of ILX23-7553, a formulation of 1,25-dihydroxy-16-ene-23-yne-vitamin D₃, a 1,25-dihydroxyvitamin D₃ analog with preclinically demonstrated antitumor and differentiating effects and diminished hypercalcemic effects. Patients and methods: The protocol consisted of five daily oral treatments during 14-day cycles at 15 dose levels from 1.3 to 45.0g/m²/day. We treated 42 heavily pretreated patients who had a variety of malignancies with 162 treatment cycles, and obtained pharmacokinetics from three patients at the two highest dose levels. Results: There were no grade 3 or 4 toxicities. Grade 1–2 toxicities included diarrhea, nausea, fatigue, constipation, and one grade 1 hypercalcemia. Average day 6 calcium was 9.26 ± 0.55mg/dl in cycle 1 and 9.30 ± 0.67mg/dl in cycle 2. Pharmacokinetics at dose levels 14 (40g/m²/day) (1 patient) and 15 (45g/m²/day) (2 patients) demonstrated an average C _max of 30.4 ± 7.8pg/ml (0.07nM) and 104 ± 38.2pg/ml (0.25nM), and AUCs of 222.5 ± 225.2pg·h/ml and 855 ± 536pgh/ml, respectively. Eight patients (19%) had stable disease. While in vitro effects have been reported at these concentrations, they were at least 10-fold lower than ED₅₀s, and the study was terminated before an MTD was reached. Conclusion: The drug is safe and has potential benefits at serum concentrations where effects begin to be noted in vitro. Further study is needed with a reformulated higher unit dose compound to determine the safety and efficacy of higher serum concentrations.