Psychotropic effects of adrenergic β-blockers on agonistic behavior between resident and intruder mice

The present study was conducted to investigate the effect of adrenergic -blockers on agonistic behavior in male mice, using quantitative ethological methods. Agonistic behavior was evoked using a resident-intruder paradigm. The following drugs were administered orally at four dose levels (vehicle, 5, 10 and 20 mg/kg) to either resident or intruder mice: dl-propranolol, practolol, d-propranolol, and l-propranolol. When the resident was treated with either dl-propranol or l-propranolol, aggressive episodes (offensive sideways posture, attack bite, tail rattle) were suppressed significantly in a dose-dependent manner, whereas practolol and d-propranolol were ineffective. All treatments except the high dose of l-propranolol failed to affect the resident's solitary behavior (locomotion). When the intruder was treated with -blockers, agonistic behavior was not altered. Since practolol does not cross the blood-brain barrier, the differential suppression of agonistic behavior is due to the central action of -blockers. d-Propranolol does cross the blood-brain barrier but is devoid of -receptor blocking property; hence l-propranolol suppression of agonistic behavior implies inactivation of brain adrenergic -receptors. The findings seem to indicate that -blockers such as dl-propranolol and l-propranolol have a psychotropic action.     

Short-term effects of herbicides on primary productivity of periphyton in lotic environments

Freshwater algae are quite sensitive to herbicides that enter running water ecosystems through direct application, aerial drift, and/or watershed run-off. However, due to a lack of suitable methodologies, few studies examine the effects of such contamination on naturally occurring attached algal communities under field conditions (i. e., exposure regimes using pulsed doses or brief episodes of peak concentrations to simulate surface run-off during storm events). This paper describes a method for determining the acute short-term effects of four herbicides (hexazinone, atrazine, tebuthiuron and metolachlor) on the net primary productivity (NPP) of periphytic algae in the field using a portable bankside incubator; NPP was measured by monitoring changes in oxygen production (mg O₂ per m²) upper surface of rock substrate per h and mg O₂ h per mg chlorophyll using the light-dark technique. All herbicides with photosynthetic inhibition as a mode of action significantly reduced NPP. The lowest observed effect concentrations (LOECs) for the herbicides were 43 g hexazinone l^–1, 109 g atrazine l^–1 and 137 g tebuthiuron l^–1. The no observed effect concentrations (NOECs) for these chemicals were <43 g hexazinone l^–1, 93 g atrazine l^–1 and 52 g tebuthiuron l^–1. Metolachlor did not significantly reduce NPP at the concentrations that were tested (range 19.6–274 g l^–1). However, community respiration (which included respiration by invertebrates) was significantly reduced at the highest metolachlor concentration (274 g l^–1). Community respiration was not significantly affected by any concentration of the other three herbicides used.     

High versus low dose granisetron,a selective 5HT3 antagonist,for the prevention of chemotherapy-induced nausea and vomiting

Summary Fifty six patients, with histologically confirmed cancer, who received highly emetogenic chemotherapy, were entered on a randomized double blind, low versus high dose, study of granisetron, a 5HT₃ receptor antagonist. A single dose of intravenous granisetron protected the majority of patients from nausea and vomiting, 160 g/kg was more effective than 40 g/kg with no more side effects. Additional doses of granisetron conferred added benefit to patients who experienced breakthrough symptoms. Granisetron at a dose range of 40–240 g/kg over a 24 hour period was well tolerated with the only side effect being mild headache.     

Biotransformation and pharmacokinetics of sulfinpyrazone (Anturan®) in man

Summary The absorption, biotransformation and elimination of sulfinpyrazone, 1,2-diphenyl-3,5-dioxo-4-(2-phenylsufinylethyl)-pyrazolidine, have been studied by administration of single 200 mg oral doses of a¹⁴C-labelled preparation to two male volunteers. Absorption from the gastro-intestinal tract was rapid and complete and the plasma concentration of unchanged drug reached maximum values of 22.67 and 13.04 µg/ml, respectively, after 1 – 2 hours. The elimination half-life in the two subjects, calculated from the decline between 3 and 8 hours, was 2.7 and 2.2 hours. The integrated concentration of unchanged sulfinpyrazone in plasma, estimated from the area under the concentration curves (AUC), was almost as high as that of total¹⁴C-substances, so the proportion of metabolized drug in plasma was low. In no case did the AUC of the three specifically determined metabolites, i.e. the sulphone G 31 442, the para-hydroxy-compound G 32 642 and the 4-hydroxy-compound GP 52 097, exceed 4% of the sulfinpyrazone value. More than 95% of whole blood radioactivity was confined to plasma. The oral dose was rapidly and completely excreted, since within 4 days more than 95% was recovered, 85% from urine and 10% from faeces. A large proportion of the dose was excreted as unchanged drug in the two volunteers: 51 and 54% of total urinary radioactivity was present as sulfinpyrazone; 8.2 and 8.8% was present as para-hydroxy-metabolite, 2.7 and 3.0% as sulphone-metabolite, and 0.6 and 0.8% as 4-hydroxy-metabolite. About 30% of urinary radioactivity consisted of highly polar metabolites. Spectroscopy of them showed that they were the C--glucuronides of sulfinpyrazone (28%) and the corresponding sulfone (2%). In these metabolites the C(4) of the pyrazolidine ring was directly attached to glucuronic acid, and thus they represent a new type of biosynthetic conjugate.     

On the Variability of Dissolution Data

Purpose. To investigate dissolution data variability and its origins. Methods. The Weibull function with four parameters, t₀ (dissolution lag-time), K (the rate parameter), (the shape parameter) and D (the fraction dissolved as t ), is used to describe the dissolution curve. The variance of the dissolution data is expressed in terms of these parameters and their individual variances ² _t0, ² _K;, ² , and ² _D. These four variances originate from variable physical properties of the dosage units and from a variable dissolution environment. Therefore, dissolution data variability depends on both, the functional form of the curve and on the variance of the physical conditions. The use of this method enables the elucidation of the sources of dissolution data variability. Results. In the case of a sigmoidal dissolution curve ( > 1), data variance is zero as dissolution begins (following dissolution lag-time). This initial variance diverges when the dissolution curve is non-sigmoidal (with < 1) but assumes a finite value, proportional to the dissolution lag-time variance (² _t0) when the data fits a regular first order rate curve ( = 1). Following a long dissolution time, data variance attains a constant value equal to the dissolution extent variance, ² _D. When the dissolution curve is sigmoidal and the variability related to the dissolution extent is sufficiently small (_D/D << 1), a maximum in the variance is expected at some intermediate time point (corresponding to the curve inflection point, when the main source of variability is dissolution lag-time t₀, or around t = 1/K + t₀, when the main sources of variability are the rate parameter K or the shape parameter ). When the curve is sigmoidal ( > 1) and the main source of variability relates to the dissolution extent, the overall variance grows with time all the way to the plateau of the dissolution curve. With a non-sigmoidal dissolution curve ( 1), data variability decreases with time soon after dissolution begins. In that case, if the main source of variability is the dissolution lag-time (t₀), the variance decreases all the way to the plateau of the dissolution curve. If the dissolution extent, D, is the main source of variability, a minimum in the variance is expected at some intermediate time point. The dissolution relative variance, on the other hand, diverges as dissolution begins and decreases with time at least until 63% of the drug is released, irrespective to the Weibull parameter values. Later, it may decrease or increase, attaining a fixed value (² _D/D²) at the plateau of the dissolution curve. Conclusions. The particular time dependence of dissolution data variance is well defined in terms of the Weibull shape parameters and their individual variances. Dissolution data variability may decrease or increase with time along the curve. It may attain a maximum or a minimum value at some intermediate time point. It may converge or diverge as dissolution begins. When the dissolution data is well fitted to the Weibull function, the sources of data variability (in terms of the Weibull parameters) may be elucidated. The variability of dissolution data originates from physical sources but is also dependent on the functional form of the curve.     

Characterization of α- and β-adrenergic receptors linked to human platelet adenylate cyclase

Summary Adrenaline and noradrenaline cause aggregation of human platelets through -adrenergic receptors, whereas isoprenaline through -adrenergic receptors can inhibit aggregation. Either type of adrenergic receptors is coupled to platelet adenylate cyclase. Stimulation and inhibition of adenylate cyclase by -and -adrenergic stimulants, respectively, had been demonstrated in human platelet lysates. These effects were characterized with regard to the effectiveness of various agonists and antagonists.Reduction of platelet adenylate cyclase activity was observed only with L-adrenaline and L-noradrenaline. This inhibitory effect, which was increased in the presence of a -adrenergic blocking agent, was half-maximal at about 1 to 2×10^–6 M adrenaline, and maximal inhibition (by 50–60%) was observed at about 3×10^–5M. Various other catecholamine and imidazoline derivatives that act as -adrenergic agonists in other cell types neither induced aggregation nor affected the enzyme activity.Adrenaline-induced inhibition of platelet adenylate cyclase was prevented by -adrenergic blocking agents. These compounds inhibited the effects of adrenaline on aggregation and on adenylate cyclase with similar efficacies. Dihydrogenated ergot alkaloids were more effective than phentolamine and yohimbine; phenoxybenzamine, tolazoline and azapetine were least effective. Adrenaline-induced inhibition of platelet adenylate cyclase was reversed by phentolamine without apparent lag phase.In the presence of -adrenergic blocking agents, adrenaline was capable of increasing adenylate cyclase activity between 20 and 50%. Only adrenaline and isoprenaline stimulated adenylate cyclase activity; other compounds that stimulate -adrenergic receptors in other cell types, including -adrenergic stimulants, had no effect on the activity of the platelet enzyme. The stimulatory effect of adrenaline was prevented by various -adrenergic blocking agents including pindolol and propranolol. Preferentially -adrenergic receptor blocking agents such as practolol and atenolol were without effect.These findings indicate that the spectrum of compounds capable of exhibiting intrinsic activity through - and -adrenergic receptors of human platelets is very narrow and that either type of platelet adrenergic receptors appears to differ from those found in other cell types.     

Influence of training dose on nicotine discrimination in humans

Non-human research indicates that drug discrimination results may depend largely on the specific training conditions, including initial training dose. It has recently been shown that humans can discriminate among different doses of nicotine delivered by nasal spray. In this study, we examined the influence of training dose on subsequent behavioral discrimination of a range of nicotine doses. Male (n=17) and female smokers (n=16) were randomly assigned to low (10 µg/kg) versus high (30 µg/kg) nicotine training dose groups and trained reliably to discriminate this dose from placebo (0) on day 1 (80% correct identification). All but six subjects (four in low, two in high) learned this discrimination and continued on to day 2, in which both groups received 0, 5, 10, 20, and 30 µg/kg in ascending order (30 min between dosings) and were tested for generalization with their training dose using quantal and quantitative behavioral discrimination tasks. Subjective responses via traditional self-report measures were also assessed. Nicotine-appropriate responding on day 2 was significantly greater in low- versus high-dose groups, especially at 5 µg/kg. However, this difference due to training dose was seen more in women than in men. Discrimination behavior was associated with subjective effects of head rush in males, and with head rush and decline in urge to smoke in females. These results show that discriminative stimulus effects of nicotine are not fixed properties of the drug, but can be influenced by training conditions, and that effects associated with this discrimination may differ between men and women.     

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.     

Comparative invstigations on the duration of action of two synthetic corticotropins

Summary The duration of the adrenocorticotrophic effects of two synthetic ACTH preparations was studied in healthy volunteers. One of the hormones, ^1–24 corticotropin, was adsorbed on zinc; the other, a D-serine¹-norleucine⁴-valinamide²⁵- ^1–25 corticotropin, was thought to be more stable, owing to the replacement of the three most susceptible amino acids in the ACTH molecule. — ^1–24 corticotropin, administered intramuscularly in a dose of 1 mg, showed biological activity lasting for 28 h, an effect equal to that which may be expected of depot preparations of pituitary extracts. D-serine¹-norleucine⁴-valinamide²⁵- ^1–25 corticotropin, administered intra muscularly in a dose of 0.16 mg, had only a weak adreno corticotrophic effect, whereas intravenously the same dose produced a greater effect that persisted for nearly 8 h. After intramuscular injection of both preparations a significant difference was found between the maximum responses shown by men and women.     

Enhancement of Solubility and Bioavailability of β-Lapachone Using Cyclodextrin Inclusion Complexes

Purpose. To explore the use of cyclodextrins (CD) to form inclusion complexes with -lapachone (-lap) to overcome solubility and bioavailability problems previously noted with this drug. Methods. Inclusion complexes between -lap and four cyclodextrins (-, -, -, and HP-CD) in aqueous solution were investigated by phase solubility studies, fluorescence, and ¹H-NMR spectroscopy. Biologic activity and bioavailability of -lap inclusion complexes were investigated by in vitro cytotoxicity studies with MCF-7 cells and by in vivo lethality studies with C57Blk/6 mice (18-20 g). Results. Phase solubility studies showed that -lap solubility increased in a linear fashion as a function of -, -, or HP-CD concentrations but not -CD. Maximum solubility of -lap was achieved at 16.0 mg/ml or 66.0 mM with HP-CD. Fluorescence and ¹H-NMR spectroscopy proved the formation of 1:1 inclusion complexes between -CD and HP-CD with -lap. Cytotoxicity assays with MCF-7 cells showed similar biologic activities of -lap in -CD or HP-CD inclusion complexes (TD₅₀ = 2.1 M). Animal studies in mice showed that the LD₅₀ value of -lap in an HP-CD inclusion complex is between 50 and 60 mg/kg. Conclusions. Complexation of -lap with HP-CD offers a major improvement in drug solubility and bioavailability.     

Functional significance of neuronal and extraneuronal transmitter uptake in rat salivary glands

Summary The effect of normetanephrine (NM) on effector cell response to noradrenaline (NA) in vivo and on extraneuronal amine uptake in vitro has been investigated in rat submaxillayr glands. Tissue slices were incubated with ³H-NA and the metabolic pattern of the radioactivity in the slices as well as of the efflux from the slices was analyzed. The level of extraneuronally retained radioactive material was found to be markedly reduced at a NM concentration of 20 g/ml after inhibition of neuronal uptake by protriptyline (PTP). In the presence of an intact neuronal membrane pump mechanism 200 g/ml of NM also reduced the level of radioactive material, indicating that NM has an inhibitory effect on extraneuronal and in higher concentration also on neuronal uptake and retention of NA and its metabolites. NM was found to markedly potentiate the secretory response to NA in vivo, when doses of NA larger than 1 g were used. At lower NA doses there was no difference between NM pretreated and control rats. When neuronal uptake was prevented by pretreatment with PTP, NM was found to potentiate the secretory response to NA over the entire dose range. Inhibition by PTP of neuronal uptake only did not affect the dose-response curve for NA and data are presented which indicate that the absence of such a potentiation is due to a decreased salivary gland blood flow induced by NA after PTP pretreatment. The increased responses recorded after NM could not be ascribed to an additive effect of this amine, since NM was found to be a very weak -receptor agonist in the rat submaxillary gland; doses of more than 1000 g i.v. were needed to detect a secretory response. The data indicate that the extraneuronal uptake of the transmitter has a functional significance in the rat submaxillary gland by reducing high transmitter concentrations in the vicinity of the receptors.     

The significance ofβ-adrenoceptor down regulation in the desipramine action in the forced swimming test

Summary The present studies were undertaken to clarify whether central-adrenoceptor down regulation is responsible for the greater effect of chronic treatment with desipramine (DMI) compared with acute treatment in the forced swimming test in rats. Repetitive administration of DMI activated the rat behaviour pattern and consequently reduced the duration of immobility. The degree of activation depended on the length of treatment, i.e. no effect when given in a single dose, moderate effect when given subchronically (3 doses) and marked activation after chronic (31 doses) treatment. Chronic treatment with DMI also produced a decrease in³H-dihydroalprenolol (³H-DHA) binding site in the cerebral cortex. Acute stimulation of brain-adrenoceptors by intracerebroventricular (i.c.v.) isoprenaline significantly, though partially, attenuated the behavioural effect of chronic DMI by ₁-adrenoceptor-related mechanisms. Similarly, chronic i.c.v. co-administration of atenolol or practolol, ₁-adrenoceptor antagonists, together with DMI attenuated both-adrenoceptor down regulation and the behavioural activation by chronic DMI. On the other hand, chronic i.c.v administration of isoprenaline, supposedly leading to down regulation of-adrenoceptors, facilitated the activating behavioural effect of DMI, as a single dose became effective. Changes, however, in³H-DHA binding parameters in the cerebral cortex were not observed after chronic isoprenaline. These results suggest that down regulation of-adrenoceptors in brain is reponsible, at least in part, for the marked activatory effect of chronic DMI in the forced swimming test, possibly by reducing an inhibitory function of ₁-adrenoceptor mediated mechanisms.     

Yohimbine diastereoisomers: Cardiovascular effects after central and peripheral application in the rat

Summary The cardiovascular effects of four yohimbine diastereoisomers, yohimbine, rauwolscine, corynanthine, and 3-epi--yohimbine, were compared in urethane-anaesthetized and conscious, normotensive Sprague-Dawley rats. Intravenous cumulative infusions (10–500 g) of the drugs to anaesthetized rats decreased blood pressure and blunted the pressor response to intravenous adrenaline injections. Corynanthine was the most potent isomer in this regard, followed by yohimbine, rauwolscine, and 3-epi--yohimbine. Depressor responses following intravenous bolus doses (40 g) showed a similar ranking. Intraventricular injections of yohimbine to anaesthetized rats decreased blood pressure dose-dependently, as did injections of corynanthine and rauwolscine. Responses indicated the ranking to be yohimbinerauwolscine>corynanthine for this effect at the 40 g dose. Heart rate was also decreased by these isomers, but not in a dose-dependent fashion. In conscious rats, the intraventricular injection of these isomers (20 g) increased blood pressure and heart rate. No differences were noted in terms of blood pressure responses; but, in causing tachycardia, the ranking was rauwolscine>yohimbine>corynanthine. These data suggest that after intraventricular application in anaesthetized rats, the effects of these alpha-adrenoceptor blockers are related to their individual affinity for the alpha 2 adrenoceptor.     

Structural and Functional Differences Between Glycosylated and Non-glycosylated Forms of Human Interferon-β (IFN-β)

Purpose. Two recombinant IFN- products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN--la) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN--lb). The structural basis for activity differences between IFN--la and IFN--lb, is determined. Methods. In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN- products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences. Results. IFN-- la was 10 times more active than IFN-- Ib with specific activities in a standard antiviral assay of 20 × 10⁷ lU/mg for IFN--la and 2 × 10⁷ lU/mg for IFN--lb. Of the known structural differences between IFN--la and IFN--lb, only glycosylation affected in vitro activity. Deglycosylation of IFN--la produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN--lb revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 × 10⁷ lU/mg). Crystallographic data for IFN--la revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure. Conclusions. Together these results suggest that the greater biological activity of IFN--la is due to a stabilizing effect of the carbohydrate on structure.     

Effects and plasma levels of propranolol and metoprolol in hyperthyroid patients

Summary The effects and plasma concentrations of different doses of propranolol and metoprolol were studied in 34 hyperthyroid patients. The initial daily doses were propranolol 160 mg or metoprolol 200 mg. If the resting heart rate remained above 75 beats per min after treatment for 4–7 days, the dose was increased and the patient re-examined after a further 4–7 days. Propranolol (n=17) caused a reduced heart rate, a decrease in serum 3,3,5-triiodothyronine (T3) and an increase in serum 3,3,5-triiodothyronine (reverse T3, rT3). In 10 patients, there was no change in T3 or rT3 until the daily dose of propranolol had been increased to 240 or 320 mg. The plasma level of propranolol was significantly correlated with the decrease in T3 and the increase in rT3. Metoprolol (n=17) caused a reduction in heart rate similar to that following propranolol. However, serum T3 was only slightly reduced even after an increase in dose to 300 or 400 mg, and serum rT3 was not altered. Metoprolol concentrations were not significantly correlated with the fall in T3. It appears that the influence of-blockers on T4 conversion is of little importance for the clinical improvement in hyperthyroid patients, and rather it is a consequence of ₁-adrenergic blockade interfering with the effect of T3. In addition, the findings support the assumption that therapeutic failure with-blockers in hyperthyroidism may be due to suboptimal treatment, and that individualized dosage is necessary.     

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.     

Effects of the calmodulin antagonists fendiline and calmidazolium on aggregation,secretion of ATP,and internal calcium in washed human platelets

Summary Ca²⁺-calmodulin dependent phosphorylation of myosin is essential for the induction of platelet shape change and subsequent reactions. Therefore, we studied the effects of the calmodulin antagonists fendiline and calmidazolium on the thrombin-induced aggregation, secretion of ATP, and increases in the intracellular free calcium concentration ([Ca²⁺]_i) in washed human platelets in the absence and presence of extracellular Ca²⁺. In Ca²⁺ free medium, fendiline (10–100 M) and calmidazolium (3–30 M) concentration-dependently inhibited aggregation. The effect of fendiline could be partly reversed by extracellular Ca²⁺ and higher thrombin concentrations. Furthermore, aggregations induced by the calcium ionophore ionomycin and by the protein kinase C-activator 4--phorbol 12-myristate 13-acetate were inhibited by fendiline, although to a smaller degree than the thrombin-induced aggregation. Thrombin-induced secretion of ATP was attenuated by low concentrations of fendiline (1–30 M) and calmidazolium (1 M) but enhanced by higher concentrations (10–30 and 3–10 M, respectively), independently of extracellular Ca²⁺. Fendiline (1–10 M) did not affect [Ca²⁺]_i in resting and thrombin-stimulated platelets. At higher concentrations (30–100 M), it induced increases in [Ca²⁺]_i in unstimulated platelets and attenuated the response to thrombin in Ca²⁺ free medium, whereas thrombin-induced Ca²⁺ influx was markedly enhanced. Similar results were obtained with calmidazolium (1–3 M). These stimulating effects on ATP secretion and on [Ca²⁺]_i of fendiline and calmidazolium may be attributed to interactions with platelet membranes by which the permeability of small cations is increased. In contrast to these actions that would be normally considered platelet-activating, our study demonstrates that the two calmodulin antagonists effectively antagonize [Ca²⁺]_i ^– mediated induction of platelet aggregation. Send offprint requests to A. Lückhoff at the above address     

The metabolism of the amyloid precursor protein and its relevance to Alzheimer's disease

Summary The pathology of Alzheimer's disease is primarily characterized by the deposition of -amyloid/A peptide as the major component of senile or neuritic plaques. The A peptide is produced as a result of proteolytic cleavage of the transmembrane protein precursor, APP, during its normal cellular metabolism. The free amino terminus of the A peptide is generated by an endopeptidic cleavage between Met⁶⁷¹-Asp⁶⁷² by a protease termed -secretase. Increased cleavage at this site takes place in a rare, inherited double mutation (Lys⁶⁷⁰-Met⁶⁷¹ to Asn⁶⁷⁰-Leu⁶⁷¹), leading to increased A production and consequent development of Alzheimer's disease on an accelerated time scale in the affected individuals, underscoring the pathological importance of -secretase activity. Cellular studies provide direct evidence that inhibition of -secretase activity would appear to be effective in inhibiting A production as a rational approach to developing therapeutics for the disease.     

Nicotine antagonizes the effects of reserpine on the striatal metabolism of dopamine, 5-hydroxytryptamine and noradrenaline in hypothermic but not in normothermic mice

Summary Our previous studies indicate that repeated nicotine administration inhibits the release of striatal dopamine in hypothermic mice. To study if similar inhibition occurs in noradrenergic and serotoninergic neurons mice were given (–)-nicotine (3 mg/kg, s.c.) repeatedly at 110, 80, 50, and 20 min before sacrifice. The interactions of nicotine with reserpine were also investigated. Reserpine (5 mg/kg, i.p.) was administered after the second nicotine dose at 60 min before sacrifice. To prevent the effects of nicotine on autonomic ganglia all mice were given hexamethonium (10 mg/kg, i.p.). Experiments were carried out at 20–22°C at which ambient temperature nicotine induced deep hypothermia or at 32–34°C to prevent the drug-induced hypothermia. The changes in striatal metabolism of dopamine, noradrenaline and 5-hydroxytryptamine (5-HT) weNicotine had temperature dependent effects on the dopamine metabolism which indicates a block of dopaminergic neurons as suggested in our earlier studies. Reserpine per se increased the homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) contents and decreased the 3-methoxytyramine (3-MT) and dopamine contents at both ambient temperatures. In hypothermic but not in normothermic, nicotine-treated mice reserpine's effect on dopamine metabolism was almost totally vanished. Nicotine and reserpine per se increased the 3-methoxy-4-hydroxyphenylethylglycol (MOPEG) content and decreased the noradrenaline content at both ambient temperatures. In hypothermic but not in normothermic mice nicotine antagonized the reserpine-induced decrease of noradrenaline content. Nicotine tended to decrease the 5-hydroxy-indoleacetic acid (5-HIAA) content in hypothermic mice but increased it in normothermic ones. Reserpine decreased the 5-HT content and increased the 5-HIAA content at both ambient temperatures. In hypothermic but not in normothermic, nicotine-treated mice the reserpine-induced effects on 5-HT metabolism were clearly reduced. These results indicate that in hypothermic mice repeatedly administered nicotine blocks the release of striatal dopamine and 5-HT and perhaps also noradrenaline. The nicotine-induced antagonism of the effects of reserpine is temperature-dependent as the nicotine-induced blockade of the neurons and thus the main mechanism behind both of these phenomena could be the sustained depolarization of the monoaminergic neurons. Send offprint requests to H. Haikala at the above address     

Antitumor activity of combretastatin-A4 phosphate, a natural product tubulin inhibitor

Summary The tubulin-binding natural product combretastatin A-4 (CA-4) was tested for antitumor activity against fresh human tumors in vitro and 2 mouse tumors, both in vitro and in vivo. In colony forming assays using 10% fetal bovine serum, CA-4 was inhibitory in 27/40 human ovary cancers with a mean IC₅₀ of 3.18 g/mL for a 1-hour exposure (n = 35 specimens) and 0.27 g/mL for a continuous exposure to CA-4 for 11–14 days (n = 5 specimens). Murine B-16 melanoma and P-388 leukemia were also highly sensitive to CA-4 in vitro with an identical IC₅₀ value of 0.0007 g/mL for continuous drug exposure for 8 days. Comparable in vitro cell culture studies performed in serum concentrations higher than 10%, revealed a significant loss of cytotoxic potency. Using the same reversed-phase HPLC technique as developed for paclitaxel, CA-4 was shown to bind to serum proteins (30,000 mw) > 99% and to albumin approximately 70%. CA-4 was only marginally active (25% increased lifespan) in DBA/2 mice bearing P-388 leukemia who were given doses of 100 mg/kg IP on either days, 1, 5 and 9 (p = 0.075 by Wilcoxon analysis) or on consecutive days 1–9 (p = 0.19 compared to control). A higher IP dose of 150 mg/kg on days 1, 5 and 9 did not delay subcutaneous B-16 melanoma tumor growth in C57/Bl mice. These findings demonstrate a substantial loss of antitumor efficacy for CA-4 in physiologic serum concentrations in vitro. No consistent antitumor activity was observed in two murine tumor models in vivo.