Varying the Unsaturation in N 4,N 9-Dioctadecanoyl Spermines: Nonviral Lipopolyamine Vectors for More Efficient Plasmid DNA Formulation

Purpose The aim of the study is to analyze the effect of varying the degree of unsaturation in synthesized N⁴,N⁹-dioctadecanoyl spermines on DNA condensation and then to compare their transfection efficiency in cell culture. Methods The N⁴,N⁹-di-C18 lipopolyamines—saturated (stearoyl), C9-cis- (oleoyl), and C9,12-di-cis- (linoleoyl)—were synthesized from the naturally occurring polyamine spermine. The ability of these novel compounds to condense DNA and form nanoparticles was studied using ethidium bromide fluorescence quenching and nanoparticle characterization techniques. Transfection efficiency was studied in several primary skin cells (FEK4, FCP4, FCP5, FCP7, and FCP8) and in an immortalized cancer cell line (HtTA) and was compared with the commercially available nonliposomal transfection formulation Transfectam^? (dioctadecylamidoglycyl spermine), which also contains two saturated C18 lipid chains. Results N⁴,N⁹-Dilinoleoyl spermine (C18, di-cis-9,12) is efficient at circular plasmid DNA (pEGFP) condensation and gives the most effective transfection in a series of primary skin cells and cancer cell lines at low charge ratios of 5.5 (± ammonium/phosphate). Conclusions The dienoic fatty acyl spermine conjugate N⁴,N⁹-dilinoleoyl spermine efficiently condenses DNA and achieves the highest transfection levels among the studied lipopolyamines in cultured cells.     

<Emphasis Type="Italic">N</Emphasis><Superscript>4</Superscript>,<Emphasis Type="Italic">N</Emphasis><Superscript>9</Superscript>-Dioleoyl Spermine Is a Novel Nonviral Lipopolyamine Vector for Plasmid DNA Formulation

Purpose To study the effect of synthesized N⁴,N⁹-dioleoyl spermine on DNA condensation and then measure its transfection efficiency in cell culture.Methods The lipopolyamine was synthesized from the naturally occurring polyamine spermine. The ability of this novel compound to condense DNA was studied using ethidium bromide fluorescence quenching and light scattering assays. Transfection efficiency was studied in primary skin cells (FEK4) and in an immortalized cancer cell line (HtTA), and compared with the commercially available transfection formulations Lipofectin and Lipofectamine.Results The synthesized N⁴,N⁹-dioleoyl spermine formula is efficient at condensing calf thymus and circular plasmid DNA and effectively transfects both primary skin cells and cancer cell lines at low charge ratios of (+/– ammonium/phosphate) 2.5.Conclusions N⁴,N⁹-Dioleoyl spermine condenses DNA and achieves high transfection levels in cultured cells.     

Very Long Chain N 4 ,N 9 -Diacyl Spermines: Non-Viral Lipopolyamine Vectors for Efficient Plasmid DNA and siRNA Delivery

Purpose

To study the effect of increasing the chain length over C-18 and varying the oxidation level in synthesized N ⁴,N ⁹-diacyl spermines on DNA and siRNA formulation, and then to compare their transfection efficiency in cell lines

Methods

The five novel very long chain N ⁴,N ⁹-diacyl polyamines: N ⁴,N ⁹-[diarachidoyl, diarachidonoyl, dieicosenoyl, dierucoyl and dinervonoyl]-1,12-diamino-4,9-diazadodecane were synthesized. The abilities of these novel compounds to condense DNA and to form nanoparticles were studied using ethidium bromide fluorescence quenching and nanoparticle characterization techniques. Transfection efficiency was studied in FEK4 primary skin cells and in an immortalized cancer cell line (HtTA), and compared with the non-liposomal transfection formulation Lipogen, N ⁴,N ⁹-dioleoyl-1,12-diamino-4,9-diazadodecane. Also, the abilities of these compounds to condense siRNA and to form nanoparticles were studied using a RiboGreen intercalation assay and their abilities to deliver siRNA into cells were studied in FEK4 and HtTA cells using fluorescein-labelled Label IT® RNAi Delivery Control, a sequenced 21-mer from Mirus.

Results

We show efficient pEGFP and siRNA formulation and delivery to primary skin and cancer cell lines.

Conclusions

Adding two C20 or C22 chains, both mono-cis-unsaturated, N ⁴,N ⁹-dieicosenoyl spermine and N ⁴,N ⁹-dierucoyl spermine, gave efficient siRNA delivery vectors, even in the presence of serum, comparable to TransIT-TKO and with excellent cell viability.     

A novel compound N,N-(Z)-N-(E)-tri-p-coumaroylspermidine isolated from Carthamus tinctorius L. and acting by serotonin transporter inhibition

Safflower, the dry flower of Carthamus tinctorius L., has long been applied for empirically treating cerebral ischemia and depression in traditional Chinese medicine. Pathogenesis of major depression involves monoaminergic transmission. The present study assessed whether safflower or its isolate would be effective in functionally regulating monoamine transporter using in vitro screening cell lines. We discovered that safflower insoluble fraction significantly inhibited serotonin uptake in Chinese hamster ovary cells stably expressing serotonin transporter (i.e. S6 cells). This fraction went through an activity-guided isolation and an active ingredient was obtained, which was subsequently elucidated as a novel coumaroylspermidine analog N¹,N⁵-(Z)-N¹⁰-(E)-tri-p-coumaroylspermidine using NMR techniques. Pharmacologically, this compound potently and selectively inhibited serotonin uptake in S6 cells or in synaptosomes, with IC₅₀ of 0.74 ± 0.15 µM for S6 cells or 1.07 ± 0.23 µM for synaptosomes and with a reversible competitive property for the 5HT-uptake inhibition. The potency of it for 5HT uptake was weaker than that of fluoxetine whereas efficacy generally similar for both. Animals treated with this testing compound showed a significant decrease in synaptosomal 5HT uptake capacity. Thus, N¹,N⁵-(Z)-N¹⁰-(E)-tri-p-coumaroylspermidine is a novel serotonin transporter inhibitor, which could improve neuropsychological disorders through regulating serotoninergic transmission.     

Synthesis of Poly(Hydroxypropylglutamine-Prazosin Carbamate) and Release Studies

Prazosin, an antihypertensive drug with postsynaptic ₁-aradrenergic blocking activity, has been coupled to poly-N ⁵-(3-hydroxypropyl-L-glutamine) (PHPG) via a carbamate linkage. PHPG was activated by p-nitrophenyl chloroformate and then reacted with prazosin to form p(HPG-prazosin carbamate) conjugate. Drug loading was 23.9% (w/w). Activated polymer and conjugates were characterized by infrared spectroscopy and differential scanning calorimetry. In vitro studies proceeded in pH 7.4 isotonic phosphate-buffered saline solution. Prazosin was released at a rate of 0.92 mg/day/100 mg conjugate from p(HPG-prazosin carbamate) particles. In vivo studies were performed with New Zealand White rabbits. P(HPG-prazosin carbamate) conjugate particles (100 mg) were suspended in 2 ml saline and injected subcutaneously into both flanks of rabbits. P(HPG-prazosin carbamate) conjugates, following an initial burst, demonstrated a nearly constant plasma prazosin concentration profile above 2 ng/ml, which was maintained for 10 days.     

Constitutive nitric oxide modulates the injurious actions of vasopressin on rat intestinal microcirculation in acute endotoxaemia

The administration of the nitric oxide (NO) synthase inhibitor, N^G-nitro-l-arginine methyl ester (L-NAME, 5 mg/kg s.c.) concurrently with Escherichia coli endotoxin (3 mg/kg i.v.) increased vascular permeability and caused mucosal damage in the rat intestine 1 h later. The vasopressin V₁ receptor antagonist, [Mca¹,Tyr(Me)²,Arg⁸]vasopressin (0.01–0.2 μg/kg s.c., 15 min before endotoxin) dose-dependently reduced this damage. These results suggest a beneficial role of NO, counteracting the injurious vascular actions of endogenous vasopressin, in maintaining intestinal mucosal integrity in acute endotoxaemic states.     

Effect of N-nitro-l-arginine on shock induced by endotoxin and by platelet activating factor in dogs

When N^G-nitro-l-arginine, a nitric oxide synthase inhibitor, administration was started 5 min prior to shock induction in anesthetized dogs, a partial restoration was observed in endotoxin-induced shock and a complete recovery in platelet activating factor (PAF)-induced shock. When N^G-nitro-l-arginine infusion was started 5 min after shock induction, no significant recovery was observed in endotoxin-induced shock and a complete recovery in PAF-induced shock. These data indicate that enhanced production of nitric oxide by vascular endothelial cells may contribute to endotoxin- or PAF-induced shock and also that some mediators including inducible nitric oxide synthase and/or cellular damage might be involved in endotoxin-induced shock.     

Tolerance and accumulation of lead by species of <Emphasis Type="Italic">Iris</Emphasis> L.

Seedling development, accumulation and distribution of lead (Pb) in Iris lactea var. chinensis (Fisch.) Koidz. and I. tectorum Maxim. were studied using plants grown in sand culture and exposed to 0–10 mmol l⁻¹ concentrations of Pb supplied as Pb(NO₃)₂ for 28 days. A significant reduction in dry weight (dw) of shoots and roots of I. lactea var. chinensis was observed at 6 and 10 mmol l⁻¹, respectively, and a significant reduction in dw of shoots and roots of I. tectorum was observed at 6 mmol l⁻¹. Concentration of Pb in the shoots and roots of I. lacteal var. chinensis exposed to 4 mmol l⁻¹ Pb reached 1,109 μg g⁻¹ and 2,408 μg g⁻¹ dw, respectively. The index of tolerance (IT) of I. lactea var. chinensis among 0–8 mmol l⁻¹ Pb treatments were not significantly different, while those of I. tectorum at 6 mmol l⁻¹ Pb were significantly decreased. The results indicated that I. lactea var. chinensis was more tolerant to Pb than I. tectorum. Sub-cellular localization of Pb in root cells was evaluated using transmission electron microscopy (TEM) and Pb deposits were found along the plasma membrane of some root tip cells of I. lactea var. chinensis treated at 10 mmol l⁻¹ Pb. Deposits of Pd were also observed along the surface, in the root tip cell wall and in the cytoplasm of a few malformed cells of I. tectorum exposed at 10 mmol l⁻¹ Pb treatment. One possible mechanism to explain these observations may be that most cells can maintain normal activities in the plant by sacrificing a small number of cells that accumulate a large amount Pb and show toxicity. Future studies should be designed to test this hypothesis.     

[125I] N6-p-hydroxyphenylisopropyladenosine,a new ligand for Ri adenosine receptors

Summary N⁶-p-Hydroxyphenylisopropyladenosine (HPIA) has been labelled with carrier-free Na[¹²⁵I] to very high specific activity (2,175 Ci/mmol) and used as an agonist ligand to characterize R_i adenosine receptors in rat cerebral cortex membranes. The binding is saturable, reversible, stereospecific and dependent on protein concentration. The specific binding at 37°C was of high affinity with an equilibrium dissociation constant K_D of 0.48 nmol/l and was saturable with 0.23 pmol of [¹²⁵I]HPIA per mg of protein. The rate constant of association, k₁, was 3.25×10⁸ l mol^–1 min^–1 and that of dissociation, k₂, 0.0110 min^–1 yielding a t_1/2 of 63 min. In competition experiments the (–)isomer of N⁶-phenylisopropyladenosine (PIA) was 16-fold more potent than the (+)isomer in competing for the binding sites. Specific binding was most effectively displaced by N⁶-cyclohexyladenosine (CHA, k_i=0.26 nmol/l), (–)PIA (k_i=0.33 nmol/l) and HPIA (k_i=0.52 nmol/l), whereas 5-N-ethylcarboxamidoadenosine (NECA, k_i-1.42 nmol/l) was less effective. The methylxanthines 3-isobutyl-1-methylxanthine (IBMX), theophylline and caffeine which have been classified as adenosine antagonists had k_i values between 5–34 mol/l. Binding of [¹²⁵I]HPIA was regulated by guanine nucleotides and divalent cations. The results indicate that [¹²⁵I]HPIA labels R_i adenosine receptors in rat brain membranes.     

Change in life cycle parameters and feeding rate of <Emphasis Type="Italic">Ceriodaphnia silvestrii</Emphasis> Daday (Crustacea,Cladocera) exposure to dietary copper

Changes in life cycle parameters (survival, growth, reproduction) and feeding rate of the tropical cladoceran Ceriodaphnia silvestrii as affected by Cu contaminated algae Pseudokirchneriella subcapitata were investigated. The dietary copper exposure ranged from 3 × 10⁻¹⁵ to 68 × 10⁻¹⁵ g Cu algal cell⁻¹. Low waterborne copper exposure (around 10⁻¹⁰ mol l⁻¹ free Cu²⁺ ions) was kept in the experiments. The results show an increasing toxic effect on C. silvestrii with copper increase in algal cells; at the highest copper exposure, all life cycle parameters were significantly affected. A concentration of 38 × 10⁻¹⁵ g Cu algal cell⁻¹ reduced egg hatching percentile and the number of neonates produced per female, but did not cause any statistically significant effect on animals survival nor to the number of eggs produced per female. The following sequence of events was observed from the lowest to the highest copper contamination: reproduction, feeding rate, body length and, at last, survival was affected. We conclude that algal cells are an important route of copper exposure and toxicity to cladocerans.     

A positron emission tomography (PET) study of cerebral dopamine D<Subscript>2</Subscript> and serotonine 5-HT<Subscript>2A</Subscript> receptor occupancy in patients treated with cyamemazine (Tercian)

Rationale Cyamemazine (Tercian) is an antipsychotic drug with anxiolytic properties. Recently, an in vitro study showed that cyamemazine possesses high affinity for serotonin 5-HT_2A receptors, which was fourfold higher than its affinity for dopamine D₂ receptors (Hameg et al. 2003).Objectives The aim of this study is to confirm these previous data in vivo in patients treated with clinically relevant doses of Tercian.Methods Eight patients received 37.5, 75, 150 or 300 mg/day of Tercian depending on their symptomatology. Dopamine D₂ and serotonin 5-HT_2A receptor occupancies (RO) were assessed at steady-state plasma levels of cyamemazine with positron emission tomography (PET), using [¹¹C]raclopride and [¹¹C]N-methyl-spiperone, respectively. The effective plasma level of the drug leading to 50% of receptor occupancy was estimated by fitting RO with plasma levels of cyamemazine at the time of the PET scan.Results Cyamemazine induced near saturation of 5-HT_2A receptors (RO=62.1–98.2%) in the frontal cortex even at low plasma levels of the drug. On the contrary, occupancy of striatal D₂ receptors increased with plasma levels, and no saturation was obtained even at high plasma levels (RO=25.2–74.9%). The effective plasma level of cyamemazine leading to 50% of D₂ receptor occupancy was fourfold higher than that for 5-HT_2A receptors. Accordingly, individual 5-HT_2A/D₂ RO ratios ranged from 1.26 to 2.68. No patients presented relevant increased prolactin levels, and only mild extrapyramidal side effects were noticed on Simpson and Angus Scale.Conclusion This in vivo binding study conducted in patients confirms previous in vitro findings indicating that cyamemazine has a higher affinity for serotonin 5-HT_2A receptors compared to dopamine D₂ receptors. In the dose range 37.5–300 mg, levels of dopamine D₂ occupancy remained below the level for motor side effects observed with typical antipsychotics and is likely to explain the low propensity of the drug to induce extrapyramidal side effects.     

A Comparison Between Polymeric Microsphere and Bacterial Vectors for Macrophage P388D1 Gene Delivery

Purpose The purpose of this study was to compare bacterial and polymeric gene delivery devices for the ability to deliver plasmid DNA to a murine macrophage P388D1 cell line. Methods An 85:15 ratio of poly(lactic-co-glycolic acid) (PLGA) and poly(β-amino ester) polymers were formulated into microspheres that physically entrapped plasmid DNA encoding for the firefly luciferase reporter gene; whereas, the same plasmid was biologically transformed into a strain of Escherichia coli engineered to produce recombinant listeriolysin O. The two delivery devices were then tested for gene delivery and dosage effects using a macrophage cell line with both assays taking advantage of a 96-well high throughput format to quantify and compare each vector type. Results Gene delivery was comparable for both vectors at higher vector dosages while lower dosages showed an improved delivery for the microsphere vectors. Delivery efficiency (defined as luciferase measurement/mg cellular protein/ng DNA delivered) was 881 luminescence mg⁻¹ ng⁻¹ for polymeric microspheres compared to 171 luminescence mg⁻¹ ng⁻¹ for the bacterial vectors. Conclusion A first head-to-head comparison between polymeric and bacterial gene delivery vectors shows a delivery advantage for polymeric microspheres that must also be evaluated in light of vector production, storage, and future potential. Saba Parsa and Yong Wang contributed equally to this work.     

Stereoselectivity in metabolism of ifosfamide by CYP3A4 and CYP2B6

The aim was to identify the hepatic cytochromes P450 (CYPs) responsible for the enantioselective metabolism of ifosfamide (IFA). The 4-hydroxylation, N²- and N³-dechloroethylation of IFA enantiomers were monitored simultaneously in the same metabolic systems using GC/MS and pseudoracemate techniques. In human and rat liver microsomes, (R)-IFA was preferentially metabolized via 4-hydroxylation, whereas its antipode was biotransformed in favour of N-dechloroethylation. CYP3A4 was the major enzyme responsible for metabolism of IFA enantiomers in human liver. The study also revealed that CYP3A (human CYP3A4/5 and rat CYP3A1/2) and CYP2B (human CYP2B6 and rat CYP2B1/2) enantioselectively mediated the 4-hydroxylation, N²- and N³-dechloroethylation of IFA. CYP3A preferentially supported the formation of (R)-4-hydroxyIFA (HOIF), (R)-N²-dechloroethylIFA (N2D) and (R)-N³-dechloroethylIFA (N3D), whereas CYP2B preferentially mediated the generation of (S)-HOIF, (S)-N2D and (S)-N3D. The enantioselective metabolism of IFA by CYP3A4 and CYP2B1 was confirmed in cDNA transfected V79 cells.     

Protective effect of N-acetylcysteine on ischaemia-induced myocardial damage in canine heart

Summary The glutathione redox pathway is an important antioxidant system in the myocardium. N-Acetylcysteine is a low molecular weight glutathione precursor that has been used clinically to replenish glutathione stores. The present study was aimed at evaluating the protective effect of N-acetylcysteine on myocardial damage resulting from permanent coronary occlusion (without reperfusion) in anaesthetized dogs. N-Acetylcysteine (150 mg kg^–1 i.v.) administered 2 min before occlusion rerduced infarct size in dogs subjected to 24 h ischemia. The infarct size as a percentage of the area at risk was 86.8 ± 3.6% (n = 11) in control (salinetreated) dogs and 68.2 ± 2.4% (n = 7; P < 0.05 vs control) in N-acetylcysteine-treated animals. Haemodynamic variables (heart rate, mean arterial pressure and ratepressure product) were similar in the control and the treated group. Regional myocardial blood flow was determined with radioactive microspheres in ischaemic and non-ischaemic zones before occlusion and 3 h post-ocelusion. N-Acetylcysteine did not influence the regional distribution of myocardial blood flow. The myocardial content of reduced glutathione was significantly (P < 0.05) decreased 3 h post-occlusion (0.53 ± 0.19 mol/ g^–1 ; n = 5) compared to either pre-occlusion values (0.94 ± 0.03 mol/g^–1; n = 8) or values 3 h post-ocelusion in sham-operated animals (0.93 ± 0.15 mo1/g^–1 ; n = 5). Depletion of myocardial glutathione 3 h post-ocelusion was not observed in dogs treated with N-acetylcysteine (0.87 ± 0.11 mol/g^–1; n = 5). Superoxide dismutase activity and malondialdehyde levels were determined in blood samples obtained from the coronary vein draining the ischaemic zone. Superoxide dismutase activity increased 10 min post-occlusion in control but not in N-acetylcysteine-treated dogs. Malondialdehyde levels were elevated in both groups after occlusion but this increase failed to reach statistical significance in the animals treated with N-acetylcysteine. This study demon strates that N-acetylcysteine treatment reduces myocardial damage after permanent coronary occlusion. The beneficial effect may be due to maintenance of myocardial glutathione and to protection against free-radicalmediated damage during the early phase of ischaemia.     

Characterization of the Human Upper Gastrointestinal Contents Under Conditions Simulating Bioavailability/Bioequivalence Studies

Purpose This study was conducted to compare the luminal composition of the upper gastrointestinal tract in the fasted and fed states in humans, with a view toward designing in vitro studies to explain/predict food effects on dosage form performance. Methods Twenty healthy human subjects received 250 mL water or 500 mL Ensure plus^? (a complete nutrient drink) through a nasogastric tube and samples were aspirated from the gastric antrum or duodenum for a period up to 3.5 h, depending on location/fluid combination. Samples were analyzed for polyethylene glycol, pH, buffer capacity, osmolality, surface tension, pepsin, total carbohydrates, total protein content, and bile salts. Results Following Ensure plus^? administration, gastric pH was elevated, buffer capacity ranged from 14 to 28 mmoL L⁻¹ ΔpH⁻¹ (vs. 7–18 mmol L⁻¹ ΔpH⁻¹), contents were hyperosmolar, gastric pepsin levels doubled, and surface tension was 30% lower than after administration of water. Post- and preprandial duodenal pH values were initially similar, but slowly decreased to 5.2 postprandially, whereas buffer capacity increased from 5.6 mmol L⁻¹ ΔpH⁻¹ (fasted) to 18–30 mmol L⁻¹ ΔpH⁻¹ (p< 0.05). Postprandial surface tension in the duodenum decreased by >30%, bile salt levels were two to four times higher, luminal contents were hyperosmotic, and the presence of peptides and sugars was confirmed. Conclusions This work shows that, in addition to already well characterized parameters (e.g., pH, and bile salt levels), significant differences in buffer capacity, surface tension, osmolality, and food components are observed pre-/postprandially. These differences should be reflected in test media to predict food effects on intralumenal performance of dosage forms.     

Reactions of vinyl chloride with RNA and DNA of various mouse tissues in vivo

The irreversible binding of ¹⁴C-vinyl chloride metabolites to RNA and DNA of mouse brain, lung, liver, kidney, spleen, pancreas, and testes after a single i.p. injection has been studied. Hydrolysates of nucleic acids from selected organs were separated on Aminex A6 for quantitation of alkylation products.Radioactivity in nucleic acids was registered in all of the studied organs with the exception of brain. RNA from spleen, pancreas and liver, and DNA from spleen and liver contained the highest amounts of radioactivity. In nucleic acids from spleen and pancreas, both organs of high metabolic activity, the entire radioactivity was found metabolically incorporated as C₁-fragments. In RNA of kidney and liver, a large part of the radioactivity was also present as incorporated C₁-fragments, but 3,N⁴-ethenocytidine (in kidney) as well as 1,N⁶-ethenoadenosine and 1,N⁶-ethenoadenine (in kidney and liver) were identified as alkylation products. In liver DNA, incorporation of C₁-fragments was insignificant, indicating different interactions of vinyl chloride metabolites (C₁-fragments and alkylating products) with RNA and DNA. The elution profiles of radioactivity in hydrolysates of liver DNA were dominated by an alkylation product of unknown structure (probably a derivative of deoxyguanosine). Possibly, 1,N⁶-ethenodeoxyadenosine and 1,N⁶-ethenoadenine were also present in liver DNA.The results are consistent with the ability of vinyl chloride to act as a multipotent carcinogen by alkylation of DNA in several tissues.     

Antagonism of discriminative stimulus effects of Δ9-THC and (R)-methanandamide in rats

Rationale In previous drug discrimination studies we observed surmountable antagonism by Δ⁹-tetrahydrocannabinol (THC) in the presence of constant doses of SR-141716 [N-(piperidin-1-yl)-5-(4-chloro-phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (0.3 and 1 mg/kg), but there was only marginal evidence for surmountable antagonism with combinations of SR-141716 and (R)-methanandamide, a chiral analog of the endocannabioid anandamide. Objective Here we examine antagonism where the cannabinoid CB1 receptor agonist [Δ⁹-THC and (R)-methanandamide] dose is held constant (i.e., the training dose) and the antagonist {i.e., SR-141716 and AM-251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; 2 ml/kg]} dose varied. We also tested the cannabinoid CB2 receptor antagonist SR-144528 {N-[(1S)-endo-1,3,3-trimethylbicyclo(2.2.1)heptan-2-yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide}. Methods Different groups of rats were trained to discriminate between vehicle and three different doses of Δ⁹-THC (1.8, 3, and 5.6 mg/kg, presumably reflecting different efficacy demands) as well as 10 mg/kg (R)-methanandamide. Dose-generalization tests involved different doses of the cannabinoid CB1 receptor agonists. Antagonist tests varied the dose of the antagonist (range: 0.1 and 3 mg/kg for SR-141716 and AM-251, and 1 to 10 mg/kg for SR-144528). Results SR-141716 and AM-251 doses dependently blocked the agonist-induced discriminative stimulus effects. SR-141716 tended to be slightly more potent than AM-251. The effective dose 50 (ED₅₀) of SR-141716 was higher in the 5.6 mg/kg Δ⁹-THC-trained group relative to the two other Δ⁹-THC-trained groups. The cannabinoid CB2 receptor antagonist SR-144528 combined with the training dose of 1.8 mg/kg Δ⁹-THC, as well as when combined with the training dose of 10 mg/kg (R)-methanandamide, did not markedly change drug-appropriate (agonist) responses. Conclusion Data support that the discriminative stimulus effects of (R)-methanandamide and its overlap with the Δ⁹-THC cue are, indeed, CB1 receptor mediated events as revealed in antagonism tests with the selective central CB1 receptor antagonists SR-141716 and AM-251. The activation of cannabinoid CB2 receptors appears to be insignificant for these discriminations.     

Enhancement of blood-brain barrier permeability to sodium fluorescein by stimulation of µ opioid receptors in mice

Summary The effects of opioids on the permeability of the blood-brain barrier (BBB) were examined in mice with sodium fluorescein as an indicator of the permeability. The brain was perfused with saline 30 min after injection of sodium fluorescein (40 mg/kg, i. v.) and examined by fluorometry. Morphine hydrochloride (0.3–10 mg/kg, s. c.) markedly increased the brain level of sodium fluorescein dose-dependently without influencing the plasma level, when administered 20 min before sodium fluorescein injection. Intracerebroventricularly (i. c. v.) injected morphine hydrochloride (0.5 and 1.0 Erg) increased the brain sodium fluorescein level. Buprenorphine (0.1 and 0.5 mg/kg, s. c.) was also effective. However, pentazocine, ethylketazocine, U-50488H and SKF-10047 had no significant influence. The i.c.v. administration of [D-Ala², McPhe⁴, Gly(ol)⁵]enkephalin (0.1 g) and [D-Ala², D-Leu⁵]enkephalin (0.5 g) but not of [D-Thr², Leu⁵]enkephalin-Thr increased the brain level of sodium fluorescein significantly. A small dose of naloxone (i. p.) significantly inhibited the effects of morphine, buprenorphine, [D-Ala², McPhe⁴, Gly(ol)⁵]enkephalin and [D-Ala³, D-Leu⁵]enkephalin. ICI-174864 co-administered i. c. v. with [D-Ala², D-Leu⁵]enkephalin was ineffective in antagonizing the effect of the latter. These findings suggest that the stimulation of µ opioid receptors results in an increase in BBB permeability to sodium fluorescein. Send offprint requests to K. Saeki     

Species variations in 5-HT3 recognition sites labeled by 3H-quipazine in the central nervous system

Summary The specific binding of ³H-quipazine to putative 5-HT₃ receptors was analyzed in multiple species. Specific and saturable binding of the radioligand could be detected in both rat (K _D = 1.2 nM; B _max = 3.0 pmol/g) and pig (K _D = 1.3 ± 0.2 nM; B _maX = 1.5 ± 0.2 p/mol/g) cortical membranes. By contrast, no significant specific binding of ³H-quipazine could be detected in human, cow, dog, turtle, mouse, guinea pig, chicken or rabbit brain membranes. These data indicate that marked species variations exist in the presence and/or density of 5-HT₃ membrane recognition sites in the central nervous system. Send offprint requests to Stephen J. Peroutka at the above address