Signaling pathways leading to phosphorylation of Akt and GSK-3β by activation of cloned human and rat cerebral D₂and D₃ receptors

Although dopamine (DA) regulates the serine/threonine kinase Akt and its downstream substrate glycogen synthase kinase-3β (GSK-3β), the direct influence of dopaminergic receptors remains poorly characterized. Short-term incubation of Chinese hamster ovary (CHO)-expressed human (h)D(?L) and hD?) receptors with DA (maximal effect, 5-10 min) phosphorylated Akt (Thr308 and Ser473) and GSK-3β (Ser9), actions blocked by the selective D? and D? antagonists, 3-[4-(4-chlorophenyl)-4-hydroxypiperidin-l-yl]methyl-1H-indole (L741,626) and (3aR,9bS)-N[4-(8-cyano-1,3a,4,9b-tetrahydro-3H-benzopyrano[3,4-c]pyrrole-2-yl)-butyl] (4-phenyl)benzamide (S33084), respectively. Similar findings were acquired with the specific D?/D? receptor agonist quinelorane, which also enhanced (10 min after administration) levels of p-Akt and p-GSK-3β in rat nucleus accumbens, an action blocked by the D?/D? receptor antagonist raclopride. Akt and GSK-3β phosphorylation mediated via CHO-expressed hD(?L) and hD? receptors was prevented by pertussis toxin and by inhibitors of insulin-like growth factor-1 receptors as well as phosphatidylinositol 3-kinase and Src. Likewise, chelation of intracellular Ca2+ and interference with an "atypical" phorbol ester-insensitive protein kinase C (PKC) abolished recruitment of Akt and GSK-3β. Inactivation of PKCμ blocked Akt and GSK-3β phosphorylation at hD(?L) receptors. However, blockade of conventional PKC isoforms attenuated the actions of DA at hD? receptors only. Furthermore, phospholipase C (PLC), calmodulin, and Akt inhibitors abolished DA-induced GSK-3β phosphorylation by hD? receptors, whereas phosphorylation by hD(?L) receptors partially involved calmodulin, Akt, and extracellular signal-regulated kinase (ERK) 1/2. In conclusion, at both hD(?L) and hD? receptors, DA elicited a G(i/o)- and Ca2+/calmodulin-dependent phosphorylation of Akt and GSK-3β via transactivation of insulin-like growth factor 1 receptor. However, significant differences were seen regarding the involvement of PLC, calmodulin, and ERK1/2.     

Analysis of multidrug effects by parameter method

目的：建立一种新的分析多药物联用效果（协同，相加或拮抗）的方法．方法：根据靶体动力学原理，引入药物等效性检验法，建立新的数学模型：Ｑ＝（Ｅｏ－Ｅｅ）／｜Ｅｅ·Ｗ－ｓｘ·Ｔ｜（Ｑ≤－１拮抗，Ｑ≥１协同，１＞Ｑ＞－１相加）其中Ｅｏ为药物联用实测效应拟合值，Ｅｅ为联用药效期望值，Ｗ为专业等效标准，大小据专业而定，一般为１０％（Ｗ＝０１）．ｓｘ为Ｅｏ和Ｅｅ共同标准误，Ｔ是单侧ｔ检验ｔ０．０５值．并设计不同实验，取得不同类型具有多重属性的数据，用此模型对实验结果进行分析，并与其他方法比较．结果：本法适用于能用Ｈｉｌ方程进行拟合的数据，质反应数据Ｅｍａｘ固定为１（１００％），不局限于联用药物是否作用于受体，联用药物数目不受限制，可对数据进行系统分析．所得结论为专业结论和统计结论的综合．结论：参数法能有效地分析多种类型联用数据     

In Vitro and In Vivo Correlation of Hepatic Fraction of Metabolism by P450 in Dogs

1-Aminobenzotriazole (ABT) has been widely used as a nonspecific mechanism-based inhibitor of cytochrome P450 (P450) enzymes. It is extensively used in preclinical studies to determine the relative contribution of oxidative metabolism mediated by P450 in vitro and in vivo. The aim of present study was to understand the translation of fraction metabolized by P450 in dog hepatocytes to in vivo using ABT, for canagliflozin, known to be cleared by P450-mediated oxidation and UDP-glucuronosyltransferases–mediated glucuronidation, and 3 drug discovery project compounds mainly cleared by hepatic metabolism. In a dog hepatocyte, intrinsic clearance assay with and without preincubation of ABT, 3 Lilly compounds exhibited a wide range of fraction metabolized by P450. Subsequent metabolite profiling in dog hepatocytes demonstrated a combination of metabolism by P450 and UDP-glucuronosyltransferases. In vivo, dogs were pretreated with 50 mg/kg ABT or vehicle at 2 h before intravenous administration of canagliflozin and Lilly compounds. The areas under the concentration-time curve (AUC) were compared for the ABT-pretreated and vehicle-pretreated groups. The measured AUC_ABT/AUC_veh ratios were correlated to fraction of metabolism by P450 in dog hepatocytes, suggesting that in vitro ABT inhibition in hepatocytes is useful to rank order compounds for in vivo fraction of metabolism assessment.     

Quantitative Analysis of Flavonoids in Scutellariae Radix of Different Sources and Seasonal Variation by HPLC

用反相ＯＤＳ柱，磷酸缓冲溶液和甲醇（６８：３２和１：１）作为流动相，对中国七个省产的黄芩和辽宁同一产地不同采收期（１９９０１９９２）黄芩中黄芩甙、黄芩素、汉黄芩甙和汉黄芩素四种主要的有效成分进行了定量分析。实验结果表明，河北承德地区野生黄芩中四种成分的含量最高。辽宁凌源野生黄芩的最佳采收期应为８月末     

Analysis of free and bound formaldehyde in squid and squid products by gas chromatography–mass spectrometry

Formaldehyde can be added illegally as a food preservative in addition to the endogenous formaldehyde that naturally occurs in aquatic products. In this study, formaldehyde was derivatized from 2,4-dinitrophenylhydrazine and analyzed using gas chromatography–mass spectrometry to investigate free and reversibly bound formaldehyde in 10 squid and squid products. The results were compared to those obtained by high-performance liquid chromatography (HPLC). The limit of detection was 2.0 mg/kg. The total concentrations of free and reversibly bound formaldehyde were, on average, higher than the free formaldehyde concentration by 26.6 mg/kg. Free formaldehyde made up, on average, 39% of total free and reversibly bound formaldehyde. The sum of the concentrations of free and reversibly bound formaldehyde was, on average, higher than the free formaldehyde concentration by 19.3 mg/kg in the HPLC method. Free formaldehyde made up an average of 39% of total free and reversibly bound formaldehyde in the HPLC method. The use of gas chromatography–mass spectrometry to detect formaldehyde in aquatic products allowed confirmation through retention time and molecular mass information. The monitoring of free formaldehyde in aquatic products and proper control of the manufacturing process could help to reduce the formaldehyde level in shredded squid products. Finally, exposure to formaldehyde from consumption of shredded squid was estimated: it was less than 0.2 mg/kg, which is the oral reference dose suggested by the US Environmental Protection Agency.     

Metabolic disorders and cardiovascular consequences of HIV infection and antiretroviral therapy

Metabolic disturbances associated with HIV disease have become an important factor in patient management and have important implications for long-term outcomes, both in regards to mortality and healthcare burden. Recent research has implicated both HIV infection itself and specific antiretroviral therapies in the development of these disorders. This review examines recent findings from research into insulin and glucose dysregulation, serum lipid abnormalities, adipose tissue and derangements in bone metabolism. This review then describes the cardiovascular consequences and management of these metabolic disorders, and summarizes current thinking on the pathogenesis and effects of antiretroviral therapy. Finally, the review raises some questions regarding ongoing challenges and unmet needs in this field of research.     

Noninvasive Monitoring of HPMA Copolymer–RGDfK Conjugates by Magnetic Resonance Imaging

Purpose  To evaluate the tumor targeting potential of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–gadolinium(Gd)–RGDfK conjugates by magnetic resonance (MR) T1-mapping. Methods  HPMA copolymers with and without RGDfK were synthesized to incorporate side chains for Gd chelation. The conjugates were characterized by their side-chain contents and r₁ relaxivity. In vitro integrin-binding affinities of polymeric conjugates were assessed via competitive cell binding assays on HUVEC endothelial cells and MDA-MB-231 breast cancer cells. In vivo MR imaging was performed on MDA-MB-231 tumor-bearing SCID mice at different time points using non-targetable and targetable polymers. The specificity of αvβ3 targeting was assessed by using non-paramagnetic targetable polymer to block αvβ3 integrins followed by injection of paramagnetic targetable polymers after 2 h. Results  The polymer conjugates showed relaxivities higher than Gd-DOTA. Endothelial cell binding studies showed that IC₅₀ values for the copolymer with RGDfK binding to αvβ3 integrin-positive HUVEC and MDA-MB-231 cells were similar to that of free peptide. Significantly lower T1 values were observed at the tumor site after 2 h using targetable conjugate (p < 0.012). In vivo blocking study showed significantly higher T1 values (p < 0.045) compared to targetable conjugate. Conclusion  These results demonstrate the potential of this conjugate as an effective targetable MR contrast agent for tumor imaging and therapy monitoring.     

Analysis of the constituents of aqueous preparations of Stachys recta by HPLC–DAD and HPLC–ESI-MS

In the present study a method based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface for the simultaneous determination of phenolic constituents in three aqueous preparations of the herbal medicinal drug Stachys recta. The developed assay was simple and effective and permitted the quality control of S. recta decoctions and infusion. Overall, 30 constituents were detected and identified, belonging mainly to three classes of compounds: caffeoylquinic acids, phenylethanol glycosides and flavonoids. 15 of them were quantified having a lower limit not less than 0.02% of the lyophilized extracts. Only seven of them were previously reported in this species, while 23 were identified for the first time as constituents of S. recta. HPLC–DAD–ESI-MS analysis provided evidence for the certain identification of the main constituents and in some cases of their isomers. Eight constituents were isolated and their structure elucidated by HPLC–ESI-MS and 1D- and 2D-NMR spectroscopy. Among the investigated preparations, the infusion seems to be the best method to extract the native constituents of the plant, while decoction is a more aggressive treatment and causes partial degradation of some acylated flavonoids.     

Adaptation of Quality by Design-Based Development of Isradipine Nanostructured–Lipid Carrier and Its Evaluation for In Vitro Gut Permeation and In Vivo Solubilization Fate

The present study demonstrated the systematic adaptation of quality by design-integrated approach for the development of novel nanostructured lipid carrier (NLC) of an anti-hypertensive drug isradipine (ISD) to address the inherent challenges such as low solubility and low oral bioavailability. Plackett-Burman design was used for preliminary screening of significant process and formulation variables (p <0.05), which were further processed using Box-Behnken design for the attainment of optimization goal that is, mean particle size (85.7 ± 7.3 nm), drug entrapment efficiency (87.4 ± 3.29%), and in vitro drug release characteristics (92.89 ± 5.47%). The optimized ISD-NLC formulation also demonstrated well-dispersed uniform-shaped particles (polydispersity index 0.207 ± 0.029), high gastrointestinal fluid stability (zeta potential ?10.17 ± 0.59 mV), and higher in vitro gut permeation (21.69 ± 2.38 μg/cm² of ISD-NLC as compared to 11.23 ± 1.74 μg/cm² in ISD suspension). Furthermore, lipolysis studies were performed for the purpose of in vivo fate, and significantly higher drug content of ISD from ISD-NLC in aqueous phase was found (72.34 ± 4.62%) as compared to drug suspension (3.01 ± 0.91%). Relative bioavailability of ISD-NLC and ISD suspension was increased by 4.2-fold and 1.78-fold in the absence and presence of cycloheximide which is a lymphatic uptake inhibitor revealing lymphatic uptake of ISD-NLC in bioavailability improvement. Hence, systematic adaptation of quality by design integrated approach improved gut permeation and potential solubilizaton fate (dynamic lipolysis) of ISD-NLC, which further improved the lymphatic uptake and biodistribution of drug thereby promisingits in vivo prospect and clinical efficacy.     

Characteristics of the cocaine-sensitive accumulation and O-methylation of 3H-(−)-noradrenaline by rabbit endometrium

Summary 1. The extraneuronal uptake and O-methylation of 2,5,6 ³H-(–)-noradrenaline was studied in segments of uterine endometrium from rabbits pretreated with 17-oestradiol and progesterone. 2. The uptake of ³H-noradrenaline was measured in MAO- and COMT-inhibited tissues and obeyed Michaelis-Menten kinetics with an apparent K _m of 78 mol/1 and a V _max of 5.4 nmol/g min. Uptake was inhibited by low Na⁺ and by potential substrates in the order dopamine > (–)adrenaline > (–)isoprenaline = 5-hydroxytryptamine. 3. Following uptake at 1.2 mol/1, efflux of ³H-noradrenaline was slow and appeared to be from two compartments, of which the first (I) had a t1/2 of 53 min and a capacity of 1.8 nmol/g. The presence of the second compartment (II) was inferred from the tissue content of ³H after 60 min of efflux, which was 3–4 times greater than predicted if the ³H was present in compartment I only. Following incubation with ³H-noradrenaline in the presence of cocaine 30 mol/1 the ³H efflux was rapid and the combined capacities of compartments I and II were greatly decreased. 4. ³H-NMN formation, measured in MAO-inhibited tissues, obeyed Michaelis-Menten kinetics with a half-saturating outside concentration of 12 mol/1 and a V _max of 0.9 nmol/g · min. The formation was inhibited by the neuronal uptake inhibitors, desipramine 3 mol/1 and metaraminol 100 mol/1 (each by 80%), but was unaffected by the extraneuronal uptake inhibitor, NMN 100 mol/1, and by oxytetracycline 100 mol/1 and methoxamine 10 mol/1. 5. ³H-NMN formation was inhibited to a small extent (by 30%) by hydrocortisone in a concentration which inhibited ³H-NMN formation in the myometrium by 84%. 6. It is concluded that the extraneuronal uptake of exogenous noradrenaline in the endometrium (a) resembles uptake in sympathetic nerves in its sensitivities to sodium ions and uptake inhibitors, and (b) resembles corticosteroid-sensitive extraneuronal uptake in that it has a low affinity for noradrenaline, but is linked with O-methylation in a fashion which renders uptake and O-methylation a relatively high-affinity, low-capacity system for removing noradrenaline.Abbreviations DOMA 3,4-dihydroxymandelic acid - DOPEG 3,4-dihydroxyphenylglycol - COMT catechol-O-methyl transferase - MAO monoamine oxidase - NMN normetanephrine - U-0521 3,4-dihydroxy-2-methyl propiophenone Send offprint requests to J. A. Kennedy at the above address     

A simple and robust quantitative analysis of retinol and retinyl palmitate using a liquid chromatographic isocratic method

Vitamin A is a vital nutritional substances that regulates biological activities including development, but is also associated with disease onset. The extent of vitamin A intake influences the retinoid content in the liver, the most important organ for the storage of vitamin A. Measurement of endogenous retinoid in biological samples is important to understand retinoid homeostasis. Here we present a reliable, highly sensitive, and robust method for the quantification of retinol and retinyl palmitate using a reverse-phase HPLC/UV isocratic method. We determined the impact of chronic dietary vitamin A on retinoid levels in livers of mice fed an AIN-93G semi-purified diet (4 IU/g) compared with an excess vitamin A diet (1000 IU/g) over a period from birth to 10 weeks of age. Coefficients of variation for intra-assays for both retinoids were less than 5%, suggesting a higher reproducibility than any other HPLC/UV gradient method. Limits of detection and quantification for retinol were 0.08 pmol, and 0.27 pmol, respectively, which are remarkably higher than previous results. Supplementation with higher doses of vitamin A over the study period significantly increased liver retinol and retinyl palmitate concentrations in adult mice. The assays described here provide a sensitive and rigorous quantification of endogenous retinol and retinyl palmitate, which can be used to help determine retinoid homeostasis in disease states, such as toxic hepatitis and liver cancer.     

The release of3H-noradrenaline by p- and m-tyramines and -octopamines,and the effect of deuterium substitution in α-position

Summary The³H-noradrenaline-releasing effects of p- and m-tyramines and -octopamines, either deuterated or not, were studied in isolated vasa deferentia of the rat (COMT inhibited and calcium-free solution in all experiments). K _m, for uptake₁ was higher for octopamines than for tyramines, but not increased by the introduction of deuterium in -position, except for (probably contaminated) deuterated p-octopamine. Other tissues were preloaded with³H-noradrenaline. After inhibition of vesicular uptake and MAO equi-releasing concentrations of the eight amines were strictly correlated withK _m, they were 6 to 7 times higher for unsubstituted octopamines than for corresponding tyramines. When only MAO (but not vesicular uptake) was inhibited, this difference decreased to about 4-fold, but the releasing potency of the deuterated amines (relative to their parent amines) remained unchanged (except for p-octopamine). When vesicular uptake and MAO were intact, unsubstituted octopamines were only 1.5 to 2.2 times less potent than the corresponding tyramines. Analysis of the efflux of³H-DOPEG confirmed that this gain in the relative potencies of octopamines is due to their increased ability to mobilize vesicular 3H-noradrenaline; moreover, deuterated amines as well were then better mobilizers than were their parent amines.It is concluded that, provided vesicular uptake is intact, the introduction of a \-OH-group enhances the ability of indirectly acting sympathomimetic amines to mobilize vesicular noradrenaline; the introduction of deuterium in -position, on the other hand, enhances this mobilizing effect exclusively when MAO is intact.Abbreviations used here COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPEG dihydroxyphenylglycol - MAO monoamine oxidase - OM-fraction column chromatographic fraction containingall O-methylated metabolites Supported by the Deutsche Forschungsgemeinschaft (SFB 176)     

Synthesis and biological evaluation of phosphonate analogues of 1α, 25-dihydroxyvitamin D3

A new series of phosphonate side chain analogues of 1alpha,25-dihydroxyvitamin D3 (1) have been synthesized. Antiproliferative activities of theses analogues (8a,b and 9a,b) using human keratinocyte cell shows that analogues which have natural A-ring show higher activity than unnatural A-ring series and almost equally active to 1alpha,25-Dihydroxyvitamin D3 (1) at 1 microM level.     

αVβ3-Targeted Delivery of Camptothecin-Encapsulated Carbon Nanotube-Cyclic RGD in 2D and 3D Cancer Cell Culture

Integrin αvβ3 is widely expressed in various types of human cancer lines and plays a key role in angiogenesis for tumor growth and metastasis. Delivery of therapeutics to αvβ3-expressing tumors can thus be a promising approach for treating cancer. For targeted delivery of anticancer therapeutics to αvβ3-expressing tumor cells, cyclic arginylglycylaspartic acid (RGD) peptide was covalently conjugated to the surface of carboxylic acid–functionalized carbon nanotubes (fCNTs), and the topoisomerase I inhibitor camptothecin (CPT) was encapsulated in the fCNTs (CPT@fCNT-RGD). CPT@fCNT-RGD was successfully delivered to αvβ3-expressing A375 cells, and compared with nontargeted CPT@fCNT, it provided 3.78- and 3.02-fold increases in the anticancer effect in 2D and 3D culture. Analysis of apoptosis-related gene expression shows that the expression levels of Bax, cleaved caspase-3, and nuclear factor kappa-light-chain-enhancer of activated B cells were significantly increased in A375 cells incubated with CPT@fCNT-RGD compared with those incubated with CPT@fCNT. These results suggest that cyclic RGD-conjugated CNTs encapsulating an anticancer therapeutic can be a promising platform for treating cancer.     

Enhancement of CD3AK cell proliferation and killing ability by α-Thujone

Thujone is a monoterpene ketone natural substance found mainly in wormwood and sage. Previous studies have shown that Thujone has various pharmacological effects, such as anti-tumor, analgesic, and insecticide. The effect of α-Thujone to human immune cells is still unknown. Our study focuses on investigating the effects and mechanism of α­Thujone to CD3AK (anti- CD3 antibody induced activated killer) cells proliferation and cytotoxicity to colon cancer cell lines. With cell proliferation and FCM assay, it is found that α­Thujone could significantly enhance CD3AK cell proliferation and expression of CD107a in a dose-dependent manner. The cytotoxicity to colon cancer cells detected by CCK-8 assay is also improved. The expressions of TNF-α and FasL detected with ELISA assay were not significantly changed. Mechanically, the study shows that α­Thujone could enhance the expression of p-ERK1/2 and p-Akt. In addition, α­Thujone has no cytotoxicity to HCT116 and SW620 cells proliferation. In a word, α­Thujone enhances CD3AK cell proliferation and cytotoxicity via the improvement of expression of CD107a, p-Akt and p-ERK1/2.     

Quantifying the Value of Orally Delivered Biologic Therapies: A Cost-Effectiveness Analysis of Oral Semaglutide

Oral semaglutide, which has undergone multiple phase 3 clinical trials, represents the first oral biologic medication for type 2 diabetes in the form of a daily capsule. It provides similar efficacy compared with its weekly injection counterpart, but it demands a dose on the order of 100 times as high and requires more frequent administration. We perform a cost effectiveness analysis using a first and second order Monte Carlo simulation to estimate quality-adjusted life expectancies associated with an oral daily capsule, oral weekly capsule, daily injection, and weekly injection of semaglutide. We conclude that the additional costs incurred to produce extra semaglutide for the oral formulation are cost effective, given the greater quality of life experienced when taking a capsule over a weekly injection. We also demonstrate that the potency of semaglutide allows the formulation to be cost effective, and less potent drugs will require increased oral bioavailability to make a cost effective oral formulation.