<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.     

Lipopolyamine-Mediated Single Nanoparticle Formation of Calf Thymus DNA Analyzed by Fluorescence Correlation Spectroscopy

Purpose The aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle formation with N⁴,N⁹-dioleoylspermine and N¹-cholesteryl spermine carbamate. Methods Fluorescence correlation spectroscopy (FCS) was used to determine the quality of ct DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen (PG) to allow fluorescence intensity fluctuation measurement and analysis. Results N⁴,N⁹-Dioleoylspermine efficiently condensed ct DNA into point-like molecules with diffusion coefficient (D) = 1.8 × 10⁻¹² m²/s and particle number (PN) = 0.7 [at ammonium/phosphate (N/P) charge ratio=1.0–1.5]. The determined PN values are close to the theoretical value of 0.6, providing evidence that the DNA conformation has been fully transformed, and thus a single nanoparticle has been detected. N¹-Cholesteryl spermine carbamate showed (slightly) poorer DNA condensation efficiency, even at higher N/P ratios (N/P = 1.5–2.5) with D = 1.3 × 10⁻¹² m²/s and PN value of 5.2. N⁴,N⁹-Dioleoylspermine is a more efficient DNA-condensing agent than N¹-cholesteryl spermine carbamate. Conclusions FCS measurement using PG as the probe is a novel analytical method to detect single nanoparticles of condensed DNA in nonviral gene therapy formulation studies.     

Design and synthesis of N4,N9-disubstituted spermines for non-viral siRNA delivery--structure-activity relationship studies of siFection efficiency versus toxicity

PURPOSE: To study the effect of sequentially changing the chain length, oxidation level, and charge distribution in N4,N9-diacyl and N4,N9-dialkyl spermines on siRNA formulation, and then to compare their lipoplex transfection efficiency in cell lines. METHODS: Eight N4,N9-diacyl polyamines: N4,N9-[didecanoyl, dilauroyl, dimyristoyl, dimyristoleoyl, dipalmitoyl, distearoyl, dioleoyl and diretinoyl]-1,12-diamino-4,9-diazadodecane were synthesized. Their abilities to bind to siRNA and form nanoparticles were studied using a RiboGreen intercalation assay and particle sizing. Two diamides were also reduced to afford tetraamines N4,N9-distearyl- and N4,N9-dioleyl-1,12-diamino-4,9-diazadodecane. Delivery of fluorescein-labelled Label IT RNAi Delivery Control was studied in FEK4 primary skin cells and in an immortalized cancer cell line (HtTA), and compared with TransIT-TKO. RESULTS: The design, synthesis, and structure-activity relationship studies of a series of N4,N9-disubstituted spermines as efficient vectors for non-viral siRNA delivery to primary skin and cancer cell lines is reported. These non-liposomal cationic lipids are promising siRNA carriers based on the naturally occurring polyamine spermine showing that C-18 is a better chain length as shorter chains are more toxic. CONCLUSIONS: N4,N9-Distearoyl spermine and N4,N9-dioleoyl spermine are efficient siRNA formulation and delivery vectors, even in the presence of serum, comparable to TransIT-TKO. However, four positive charges distributed as in spermine was significantly more toxic.     

VKORC1-3673G>A、CYP2C9*3、CYP4F2 rs2108622和CYP2C19*2基因多态性对中国汉族房颤患者华法林维持剂量的影响

目的 探讨VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622和CYP2C19^*2位点基因多态性对中国汉族房颤患者华法林维持剂量的影响。方法 收集107例服用华法林达维持剂量的汉族房颤患者的血样和临床相关资料,应用PCR-RFLP法检测VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622和CYP2C19^*2基因型,采用独立样本t检验分析基因型与华法林维持剂量的相关性。多元线性回归建立给药模型,探讨基因多态性对华法林维持剂量的影响。结果 VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622基因多态性和患者年龄、体质量能解释45.2%的华法林维持剂量差异。CYP2C19^*2基因多态性对本研究人群华法林维持剂量无影响。结论 VKORC1-3673G>A、CYP2C9^*3、CYP4F2 rs2108622基因多态性显著影响中国汉族房颤患者的华法林维持剂量。     

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.     

Formation of 3,N4-ethenocytidine moieties in RNA by vinyl chloride metabolites in vitro and in vivo

Rats were exposed to [1,2-¹⁴C] vinyl chloride. Liver RNA was isolated, hydrolyzed, and the nucleosides separated on Aminex-A-6. Besides the physiological bases and 1,N⁶-ethenoadenosine, radioactivity was also incorporated into 3,N⁴-ethenocytidine. Radioactive 3,N⁴-ethenocytidine moieties were also formed on incubation of polycytidylic acid with rat liver microsomes, NADPH and [¹⁴C] vinyl chloride. These alkylation mechanisms are consistent with the mutagenic and cancerogenic properties of vinyl chloride.

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Zusammenfassung RNS wurde aus der Leber von Ratten, die einer Atmosphäre mit [1,2-¹⁴C] Vinylchlorid ausgesetzt waren, isoliert. Nach enzymatischer Hydrolyse und Trennung der Nukleoside auf Aminex-A-6 wurde außer den physiologischen Nukleosiden und dem bereits beschriebenen 1,N⁶-Äthenoadenosin auch ein Radioaktivitätseinbau in 3,N⁴-Äthenocytidin gefunden. Eine Bildung von 3,N⁴-Äthenocytidinresten wurde auch beobachtet, wenn Polycytidylsäure, Rattenlebermikrosomen, NADPH-regenerierendes System und [¹⁴C] Vinylchlorid inkubiert wurden.

     

N-(4-甲氧羰基-4-邻苯二甲酰亚氨基丁酰基)-N-取代甘氨酸、脯氨酸和焦谷氨酸的合成

报道11个预期有血管紧张素转化酶抑制活性的N-(4-甲氧羰基-4-邻苯二甲酰亚氨基丁酰基)-N-取代甘氨酸(VII_1～9)、脯氨酸(VII₁₀)和焦谷氨酸(VIl₁₁)的合成和鉴定。所有上述化合物以及与VⅡ_1～9相应的叔丁酯(VI_1～9)均未见文献报道。药理初试结果显示，化合物VII₈，VII₉和VI₁₀均有明显降压活性。     

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.     

Atomoxetine occupies the norepinephrine transporter in a dose-dependent fashion: a PET study in nonhuman primate brain using (S,S)-[18F]FMeNER-D2

Rationale Atomoxetine is a potent and selective norepinephrine transporter (NET) reuptake inhibitor acting as a nonstimulant for the treatment of attention-deficit/hyperactivity disorder (ADHD). Previous positron emission tomography (PET) studies had failed to demonstrate the feasibility of measuring a dose-dependent and saturable NET occupancy in human brain using [¹¹C]MeNER.Objectives To determine if atomoxetine occupies NET in a dose-dependent fashion using (S,S)-[¹⁸F]FMeNER-D₂ in nonhuman primate brain.Methods A total of eight PET measurements were performed in two cynomolgus monkeys. Each monkey was examined four times with PET: under baseline conditions and after steady-state infusion with 0.03, 0.06, or 0.12 mg/kg/h of atomoxetine. A prolonged intravenous (i.v.) infusion design was developed rather than an i.v. bolus to better mimic an oral absorption profile and to reach plasma steady state.Results During baseline conditions, (S,S)-[¹⁸F]FMeNER-D₂ uptake was highest in the locus coeruleus, thalamus, mesencephalon, and the cingulate gyrus, whereas the radioactivity in the caudate was low. Peak equilibrium measurements were achieved using (S,S)-[¹⁸F]FMeNER-D₂ in contrast to the previously reported data for [¹¹C]MeNER. After administration of atomoxetine, a dose-dependent occupancy from 38 to 82% was observed for various brain regions known to contain high densities of NET.Conclusions This is the first in vivo PET study to successfully demonstrate the ability to measure a dose-dependent change in NET occupancy in brain using (S,S)-[¹⁸F]FMeNER-D₂. Furthermore, an asymptotic relationship between N-desmethylatomoxetine plasma concentration and NET occupancy was established. In total, these data encourage further PET studies using (S,S)-[¹⁸F]FMeNER-D₂ in humans.     

Higher levels of HIV DNA in memory and naive CD4 T cell subsets of viremic compared to non-viremic patients after 18 and 24 months of HAART

The degree of infection of memory and naive CD4⁺ T cells in patients treated with HAART and with durable undetectable or detectable viral load in plasma was evaluated. The following two groups of patients were analyzed cross-sectionally: (i) patients with undetectable HIV RNA plasma levels during follow-up (responders); (ii) patients with no reduction or with rebound in HIV RNA levels during treatment (non-responders). Patients were examined following 6, 12, 18 and 24 months of HAART, respectively, by quantifying: (i) plasma HIV RNA load; (ii) CD4⁺ T cells; (iii) memory and naive CD4⁺ T cells; (iv) HIV DNA levels in memory and naive CD4⁺ T cells. HIV RNA plasma levels were significantly higher in non-responders vs responders at each time point (P<0.02), while CD4⁺ T cell counts as well as memory and naive CD4⁺ T cell levels were comparable in both viremic and non-viremic patients. However, higher HIV DNA values were observed in both memory and naive CD4⁺ T cells of non-responders vs responders after 18 and 24 months of HAART (P<0.02), suggesting an increased amount of HIV-infected naive CD4⁺ T cells and a sustained high degree of infection of memory CD4⁺ T cells. Immunological reconstitution following HAART might potentially be hampered in viremic patients despite the absolute increase in CD4⁺ T cell counts.     

Circadian variation in the head twitch response produced by 5-methoxy-N1,N1-dimethyltryptamine and p-chloroamphetamine in the mouse

Circadian fluctuations were measured in the head twitch response produced by 5-methoxy-N¹,N¹-dimethyltryptamine (5-MeODMT) and p-chloroamphetamine (PCA) in male BK. TO mice. The effects of depleting brain 5-hydroxytryptamine (5-HT) with p-chlorophenylalanine (PCPA) on the 5-MeODMT in the mouse were also studied. Changes in brain 5-HT and 5-hydroxyindoleacetic (5-HIAA) were concomitantly determined. PCPA (400 mg/kg IP twice on consecutive days) significantly increased the number of head twitches induced by 5-MeODMT (5 mg/kg IV) on days 3 and 5 after the initial injection of PCPA when 5-HT and 5-HIAA were also significantly reduced. On day 12, there was no significant difference in the number of head twitches between mice administered PCPA and those given saline, and 5-HT and 5-HIAA levels were nearly back to normal. PCPA, using the same dose schedule, significantly reduced the number of head twitches induced by PCA when PCA was administered 24h after the second injection of PCPA (day 3). Mice maintained on a 12-h light-dark cycle showed a maximum response to the direct 5-HT receptor agonist 5-MeODMT (5 mg/kg IV) towards the end of the dark period, when the 5-HT level was at its lowest. p-Chloroamphetamine, which causes release of 5-HT from pre-synaptic neurones, produced a peak head twitch response in the middle of the light period when 5-HT and 5-HIAA levels were maximal, while the response towards the end of the dark period was significantly less than that at other tines tested. It is concluded that 5-HT receptor response shows a circadian rhythm related to both pre-synaptic availability of 5-HT and post-synaptic receptor sensitivity.     

Cytotoxic activity and cellular processing in human ovarian carcinoma cell lines of a new platinum(II) compound containing a fluorescent substituted propylene diamine ligand

A new fluorescent platinum(II) compound containing the N,N′-bis-(anthracen-9-ylmethyl)propane-1,3-diamine as a carrier ligand has been designed, synthesized and characterized. High cytotoxic activity of cis-[Pt(bapda)Cl₂] is observed in A2780 and A2780R cells (human ovarian carcinoma sensitive and cisplatin-resistant, respectively). Nevertheless, cross-resistance to platinum from cis-[Pt(bapda)Cl₂] in the A2780R cells was found. To study the role of GSH towards inactivation of cis-[Pt(bapda)Cl₂], GSH-depleted and non-depleted A2780R cells were used in several in vitro studies. The results suggest that cis-[Pt(bapda)Cl₂] is not susceptible to the inactivation by GSH. Cellular processing of bapda and cis-[Pt(bapda)Cl₂] was followed using fluorescence microscopy in the A2780, the A2780R and GSH-depleted A2780R cells. Interestingly, differences in the cellular processing followed by fluorescence microscopy between normal and GSH-depleted A2780R cells have been observed for the carrier ligand. Sequestration of these compounds in acidic lysosomes is visible after incubation in most cases, and no fluorescence was observed in the nucleus. Interaction of cis-[Pt(bapda)Cl₂] with calf thymus DNA strongly suggests that the this new platinum(II) compound intercalates between the DNA base pairs. Additionally, the reaction of cis-[Pt(bapda)Cl₂] with 9-ethylguanine appears to be very slow, as studied by ¹H and ¹⁹⁵Pt NMR spectroscopy.     

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.     

Lipid microencapsulation in starch

Short microwave heating of granular potato, waxy corn and tapioca starches with such lipids as cis-9-octadecenoic acid (oleic acid), cis,cis-9,12-octadecadienoic acid (linoleic acid), octadecanoic acid (stearic acid), ethyl cis-9-octadecenoate, ethyl cis,cis-9,12-octadecadienoate and methyl octadecanoate provided microcapsules in which encapsulated guest molecules did not interact with starch microcapsules. On the formation of microcapsules, the lipid guest molecules did not react to starches. The encapsulation yield varied between almost 11–94%.     

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.     

7α-和7β-甲基-10β,17β-二乙酰氧基-△4-雌甾烯-3酮的抗早孕作用

7α-和7β-甲基-10β,17β-二乙酰氧基-△⁴-雌甾烯-3酮(简称7α-和7β-甲-乙氧雌酮)对小鼠抗早孕ED₅₀分别为1.6和5.5 mg/kg。7α-甲-乙氧雌酮在大鼠也有抗早孕作用并使血浆孕酮浓度降低,应用10 μg/ml浓度能抑制离体妊娠大鼠卵巢孕酮合成。7α-和7β-甲-乙氧雌酮与兔子宫胞浆雌二醇受体的相对结合亲和力(RBA)分别为10.8和1.5,与孕酮受体的RBA均<1.7α-和7β-甲-乙氧雌酮都有较弱的雌激素和抗雌激素活性。     

S-adenosyl- -methionine N-ole-1-oyltaurate: pharmacokinetic of the orally administered salt in rats

A pharmacokinetic study based on the distribution of radioactivity from the double labelled S-adenosyl- -methionine (SAM) has been carried out by oral administration of the liposoluble stable salt [methyl-¹⁴C, 8-³H]SAM N-ole-1-oyltaurate to rats. The SAM sulfate p-toluensulfonate salt, the only SAM salt at present commercialized as drug, was chosen as reference compound to have a comparative pharmacokinetic analysis. The metabolism of the SAM is characterised by a differential use of the two labelled moieties by the various organs, liver being the most active in metabolizing the sulfonium compound with a preferential uptake of the methyl-¹⁴C fragment. The radioactivity detected after the administration of [methyl-¹⁴C, 8-³H]SAM N-ole-1-oyltaurate is, in all the organs examined, two times higher than the [methyl-¹⁴C, 8-³H]SAM sulfate p-toluensulfonate compound, attesting that the liposoluble [methyl-¹⁴C, 8-³H]SAM N-ole-1-oyltaurate is provided with better bioavailability.     

Determination of the adenosine A1 agonist N-cyclopentyladenosine in rat blood by solid-phase extraction and HPLC

A solid-phase extraction procedure has been developed for the isolation of the adenosine A1 receptor agonist N⁶-cyclopentyladenosine from rat blood. The biological samples were spiked with N⁶-cyclopentyladenosine and the analogue N⁶-cyclohexladenosine (internal standard), diluted with sodium hydroxide, loaded onto disposable cartridges with subsequent desorption with methanol and analysis by HPLC. The performance of columns pre-packed with different C18-bonded silica phases or with a polymeric reversed-phase sorbent (Oasis HLB) was assessed. The highest extraction efficiencies (recovery rates>83.3%) for the two N6-alkyl substituted adenosines were achieved by the Oasis HLB cartridges. In addition, the polymeric sorbent provided reproducible recoveries (relative standard deviation<4.8%), whereas large variations (relative standard deviation values, 9–16.3%) in the extraction yields were observed using the conventional silica-based C18 cartridges. The described sample preparation method is rapid, simple, selective and it is suitable for pharmacokinetic studies.