~(153)Sm-EDTMP联合~(99m)Tc亚甲基二膦酸盐治疗多发性骨转移癌的临床观察

目的　评价 1 53钐 -乙二胺四亚甲基膦酸盐 (1 53Sm- EDTMP)与 99m Tc亚甲基二膦酸盐 (商品名云克 )联合应用治疗多发性骨转移癌的疗效。方法　各种骨转移癌 132例 ,随机分组治疗 ,第 1组 4 2例患者仅静脉注射 1 53Sm- EDTMP,1.0× 10 7Bq/ kg,第 2组 5 0例患者 1 53Sm- EDTMP联合 99m Tc亚甲基二膦酸盐治疗 ,1 53Sm- EDTMP用量不变 ,并与静脉注射 1 53Sm- EDTMP后的第 5天开始用 99m Tc亚甲基二膦酸盐连续 5 d。第 3组 4 0例患者静脉滴注博宁 ,10 d1个疗程。结果　第 1组镇痛有效率为 6 1% ,疼痛缓解持续时间平均 4周。第 2组联合用药组镇痛有效率达 80 % ,疼痛缓解持续时间平均 8周。第 3组镇痛有效率 4 2 % ,疼痛缓解持续时间平均 2周。 3组疗效有显著统计学意义 (P<0 .0 5 )。结论　不论 1 53Sm - EDTMP单独使用还是与 99m Tc亚甲基二膦酸盐联合用药都是治疗多发骨转移的有效止痛方法 ,相比之下 ,联合用药可明显提高镇痛效率。     

稠环磺酰胺衍生物的合成与抗菌活性研究

为了寻找抗菌候选化合物,在前期研究基础上,18个稠环磺酰胺衍生物被设计合成,经1H NMR、13C NMR和MS确认结构。采用两倍稀释法对目标物进行体外抗菌活性测试,结果表明:该类衍生物对所测细菌有不同程度的抑制活性,尤以化合物Ⅱi、Ⅱr的抗菌活性最为突出,其中前者对金葡菌(S.aureus)、大肠埃希菌(E.coli)和耐甲氧西林金葡菌(MRSA)的最小抑菌浓度(MIC)分别为8、32和16μg·mL^-1,后者对S.aureus、E.coli及MRSA的MIC分别为8、64和32μg·mL^-1,两者的抗MRSA活性较显著,值得进一步结构优化和深入研究。     

Neuroanatomy and neurochemistry of rat cornea: Changes with age

PurposeTo characterize the entire rat corneal nerve architecture, the changes that occur with aging, and its sensory, sympathetic, and parasympathetic fiber distribution.MethodsSprague-Dawley rats (aged 1 day to 2 years old) of both sexes were euthanized, and the whole corneas were immunostained with protein gene product 9.5 (PGP9.5). The specimens were double-labeled with antibodies against calcitonin gene-related peptide (CGRP) and substance P (SP) as sensory nerve markers, vasoactive intestinal peptide (VIP) as a parasympathetic nerve marker, and neuropeptide Y (NPY) and tyrosine hydroxylase (TH) as markers of sympathetic fibers. Relative nerve density positive for each antibody was assessed by computer-assisted image analysis.ResultsThick nerve trunks enter the cornea in the middle of the stroma and run towards the anterior stroma, subsequently dividing into smaller branches that penetrate upwards into the epithelium to form the subbasal nerve bundles. There was no significant difference in corneal innervation between sexes. CGRP and SP were the major sensory neuropeptides with 47.6% ± 3.5% and 34.9% ± 5.1%, respectively, of the total nerves. VIP was 18.4% ± 5.7%, and NPY and TH positive fibers took up 6.92% ± 2.66% and 2.92% ± 1.52%, respectively. Epithelial nerve density increased with age, reached full development at 5 weeks, and decreased at 120 weeks.ConclusionThis study provides a complete nerve architecture and content of components of sensory, parasympathetic, and sympathetic nerves in the rat cornea. The normal innervation pattern described here will provide an essential baseline for investigators who use the rat model for assessing corneal pathologies that involve nerve alterations.     

α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction

β-Phosphoglucomutase (βPGM) catalyzes isomerization of β-d-glucose 1-phosphate (βG1P) into d-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a β-d-glucose 1,6-bisphosphate (βG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of βG1P deliver novel step 1 transition state analog (TSA) complexes for βPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the β-d-glucopyranose ring in the βG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O–P bond orientation for binding the phosphate in the inert phosphate site differs by ∼30° between steps 1 and 2. By contrast, the orientations for the axial O–Mg–O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of βG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.Efficient enzyme catalysis of the manipulation of phosphates is one of the great achievements of evolution (1). Enzymes that operate on phosphate monoesters and anhydrides transfer the phosphoryl moiety, PO₃⁻, with rate accelerations approaching 10²¹ for monoesters, placing them among the most proficient of all enzymes (1). Phosphomutases, including α-phosphoglucomutase (αPGM) (2, 3) and β-phosphoglucomutase (βPGM) (4–6), phosphoglycerate mutase (7), α-phosphomannomutase (αPMM/PGM) (8), and N-acetylglucosamine-phosphate mutase (9), merit special attention because these enzymes have to be effective in donating a phosphoryl group to either of two hydroxyl groups that have intrinsically different reactivity. Only when both half-reactions of a phosphomutase are accessible to mechanistic analysis can the problem of how an enzyme accommodates two distinct chemistries within a single active site be resolved. Hexose 1-phosphate mutases, including enzymes central to glycolysis and other metabolic pathways, are well characterized (10, 11). They are generally activated by phosphorylation to form a covalent phosphoenzyme, which then donates its PO₃⁻ group to either of its substrates to deliver a common, transient, hexose 1,6-bisphosphate intermediate species. However, structural studies on phosphomutases are complicated by the rapid and often imbalanced equilibrium position between the substrates, and kinetic studies are problematic because of competitive, parallel pathways of enzyme activation and substrate inhibition (12, 13). As a result, transition states (TSs) for both half-reactions have not hitherto been accessible for mechanistic analysis.βPGM is the best-characterized hexose 1-phosphate mutase and is a member of the haloacid dehalogenase (HAD) superfamily (14), which has 58 HAD homologs in Homo sapiens (11). The key cellular role for βPGM is to support growth on maltose (14), which demands isomerization of β-d-glucose 1-phosphate (βG1P) via β-d-glucose 1,6-bisphosphate (βG16BP) into d-glucose 6-phosphate (G6P), a universal source of cellular energy. This interconversion is achieved via a transient, covalent phosphoenzyme intermediate involving an essential aspartic acid, Asp8, to conserve the phosphoryl group that migrates intermolecularly (Fig. 1). Mechanistically, this pathway demands the architecture of the catalytic site to be effective in promoting phosphoryl transfer from phospho-Asp8 to the 6-OH group of βG1P (step 1), followed by reverse phosphoryl transfer from 1β-OH of βG16BP to Asp8 (step 2).Open in a separate windowFig. 1.Reaction scheme and free energy profile for the conversion of βG1P into G6P via βG16BP catalyzed by βPGM. The phosphoryl transfer reaction between βG1P and the phosphoenzyme (βPGM^P) is step 1 (transferring phosphate is shown in blue), and the equivalent reaction between G6P and the phosphoenzyme is step 2 (transferring phosphate is shown in red). The two intermediate complexes are labeled βG16BP and βG61BP to indicate the two orientations of bound β-bisphosphoglucose. Intramolecular hydrogen bonds within the glucose phosphates are indicated in green. The PDB ID codes (shown in brown) for the structures of metal fluoride ground state analog (GSA) and TSA complexes are listed next to the corresponding steps. G6P is ca. 8 kJ⋅mol⁻¹ lower in free energy than βG1P at equilibrium (12). βG1P binds fivefold less tightly than G6P in an AlF₄⁻ TSA complex, corresponding to a binding energy difference of ca. 4 kJ⋅mol⁻¹. This places the TSA for step 1 (blue) ca. 12 kJ⋅mol⁻¹ (4 kJ⋅mol⁻¹ + 8 kJ⋅mol⁻¹) higher in free energy than the TSA for step 2 (red). The free energy levels of TS1 and TS2 are placed only approximately, using the assumption that the free energy difference (wavy arrows) between the TSA complex and the true TS is similar for both step 1 and step 2. The approximate relative free energy levels for the intermediate enzyme-bound states denoted with βG16BP and βG61BP are based on published data (13).Step 2 has been studied intensively, with analyses focused on structural studies of trifluoromagnesate (MgF₃⁻) and tetrafluoroaluminate (AlF₄⁻) transition state analogs (TSAs) and trifluoroberyllate ground state analogs for G6P complexes (4–6, 15). ¹⁹F NMR resonances for these complexes additionally have provided in situ probes for the electronic and protonic environment of the phosphate moiety in the active site (4–6, 15, 16). Such studies have confirmed a trigonal bipyramidal (tbp) TS associated with inline stereochemistry and general acid–base catalysis, following the rearrangement of near-attack conformers (6). By contrast, step 1, involving phosphorylation of the 6-OH group of βG1P, is not well understood. The corresponding TSA complexes hitherto have proved inaccessible; attempted crystallization of the mutase using βG1P with magnesium and fluoride provides the same MgF₃⁻ TSA complex as is formed directly with G6P because residual enzyme activity catalyzes mutation of βG1P into G6P at a rate competitive with crystallization of the complex (17). Similarly, although ¹⁹F NMR studies have identified a transient TSA complex for an AlF₄⁻ complex of βG1P, it readily isomerizes into the corresponding TSA complex of G6P (SI Appendix, Fig. S1). This impasse is resolved here by the synthesis and use of stable analogs of βG1P that resist mutase-catalyzed isomerization. Because it has been established that α-fluorination of 6-phosphonomethyl-6-deoxy-glucose (G6CP) can enhance or impair analog binding to glucose 6-phosphate dehydrogenase, depending on the stereochemistry of the α-fluorine substituent (18), we have synthesized both diastereoisomeric α-monofluoromethylenephosphonate analogs of βG1P, its methylenephosphonate analog, and the three corresponding phosphonate 1α-hydroxyl analogs. We have identified the two best-binding analogs by ¹⁹F NMR and measured their affinities with βPGM in TSA complexes using fluorescence titration. We have thereby obtained three high-resolution crystal structures of TSA complexes for step 1 of the mutase catalytic reaction. Their comparison with TSA complexes for step 2 establishes the substantially different binding modes for βG1P and G6P in their respective reactions.     

Aryl Ester Prodrugs of Cyclic HPMPC. I: Physicochemical Characterization and In Vitro Biological Stability

Purpose. The chemical, enzymatic, and biological stabilities and physical properties of a series of salicylate and aryl ester prodrugs of the antiviral agent, cyclic HPMPC, were evaluated to support the selection of a lead compound for clinical development. Methods. Chemical stabilities of the prodrugs in buffered solutions at 37°C were determined. Stability was also studied in the presence of porcine liver carboxyesterases (PLCE) at pH 7.4 and 25°C. Tissue stabilities were examined in both human and dog intestinal homogenates, plasmas and liver homogenates. Prodrug and product concentrations were determined by reverse phase HPLC. Results. Chemical degradation of the prodrugs resulted in the formation of both cyclic HPMPC and the corresponding HPMPC monoester. Chemical stability was dependent on the orientation of the exo-cyclic ligand; the equatorial isomers were 5.4- to 9.4-fold more reactive than the axial isomers. In the presence of PLCE, the salicylate prodrugs cleaved exclusively to give cyclic HPMPC and not the HPMPC monoester. In plasma, but not intestinal or liver homogenates, the salicylate esters of cyclic HPMPC cleaved readily with a rate dependent on the chain length of the alkyl ester substituent. Conclusions. The carboxylate function on the salicylate prodrugs of cyclic HPMPC provides an additional handle to chemically modify the lipophilicity, solubility and the biological reactivity of the prodrug. In tissue and enzymatic studies, the major degradation product is cyclic HPMPC. The salicylate ester prodrugs are attractive drug candidates for further in vivo evaluation.     

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.     

Secretion of antiretroviral chemokines by human cells cultured with acyclic nucleoside phosphonates

Acyclic nucleoside phosphonates are novel class of clinically broadly used antivirotics effective against replication of both DNA viruses and retroviruses including human immunodeficiency virus (HIV). We have investigated their in vitro effects on immune defence mechanisms in human peripheral blood mononuclear cells, with the main emphasis on expression of cytokines which are able to suppress the entry of HIV in cells. Included in the study were prototype acyclic nucleoside phosphonates, i.e. 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; adefovir), 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP), (R)-and (S)-enantiomers of 9-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; tenofovir] and [(S)-PMPA], and of 9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine [(R)-PMPDAP] and [(S)-PMPDAP], and their N(6)-substituted derivatives. Some of the compounds were found to substantially enhance secretion of chemokines such as macrophage inflammatory protein-1alpha (MIP-alpha/CCL3), and "regulated on activation of normal T cell expressed and secreted" (RANTES/CCL5). Secretion of MIP-1beta/CCL4 was only marginally increased, whereas production of interleukin-16 (IL-16) and interferon-gamma (IFN-gamma) remained uninfluenced. The most effective proved to be the N(6)-cyclooctyl-PMEDAP, N(6)-isobutyl-PMEDAP, N(6)-pyrrolidino-PMEDAP, N(6)-cyclopropyl-(R)-PMPDAP, and N(6)-cyclopentyl-(R)-PMPDAP derivatives. Remarkably enhanced secretion of chemokines was reached within 2-4 h of the cell culture, and was observed at concentration of 2-5 microM. It may be suggested that acyclic nucleoside phosphonates represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties.     

Phosphorus-based Flame Retardancy Mechanisms—Old Hat or a Starting Point for Future Development?

Different kinds of additive and reactive flame retardants containing phosphorus are increasingly successful as halogen-free alternatives for various polymeric materials and applications. Phosphorus can act in the condensed phase by enhancing charring, yielding intumescence, or through inorganic glass formation; and in the gas phase through flame inhibition. Occurrence and efficiency depend, not only on the flame retardant itself, but also on its interaction with pyrolysing polymeric material and additives. Flame retardancy is sensitive to modification of the flame retardant, the use of synergists/adjuvants, and changes to the polymeric material. A detailed understanding facilitates the launch of tailored and targeted development.     

Gallium(III) complexes of NOTA‐bis (phosphonate) conjugates as PET radiotracers for bone imaging

Ligands with geminal bis(phosphonic acid) appended to 1,4,7‐triazacyclonone‐1,4‐diacetic acid fragment through acetamide (NOTAM^BP) or methylenephosphinate (NO2AP^BP) spacers designed for ⁶⁸Ga were prepared. Ga^III complexation is much faster for ligand with methylenephosphinate spacer than that with acetamide one, in both chemical (high reactant concentrations) and radiolabeling studies with no‐carrier‐added ⁶⁸Ga. For both ligands, formation of Ga^III complex was slower than that with NOTA owing to the strong out‐of‐cage binding of bis(phosphonate) group. Radiolabeling was efficient and fast only above 60 °C and in a narrow acidity region (pH ~3). At higher temperature, hydrolysis of amide bond of the carboxamide‐bis(phosphonate) conjugate was observed during complexation reaction leading to Ga–NOTA complex. In vitro sorption studies confirmed effective binding of the ⁶⁸Ga complexes to hydroxyapatite being comparable with that found for common bis(phosphonate) drugs such as pamindronate. Selective bone uptake was confirmed in healthy rats by biodistribution studies ex vivo and by positron emission tomography imaging in vivo. Bone uptake was very high, with SUV (standardized uptake value) of 6.19 ± 1.27 for [⁶⁸Ga]NO2AP^BP) at 60 min p.i., which is superior to uptake of ⁶⁸Ga–DOTA‐based bis(phosphonates) and [¹⁸F]NaF reported earlier (SUV of 4.63 ± 0.38 and SUV of 4.87 ± 0.32 for [⁶⁸Ga]DO3AP^BP and [¹⁸F]NaF, respectively, at 60 min p.i.). Coincidently, accumulation in soft tissue is generally low (e.g. for kidneys SUV of 0.26 ± 0.09 for [⁶⁸Ga]NO2AP^BP at 60 min p.i.), revealing the new ⁶⁸Ga complexes as ideal tracers for noninvasive, fast and quantitative imaging of calcified tissue and for metastatic lesions using PET or PET/CT. Copyright © 2014 John Wiley & Sons, Ltd.     

Assessment of a gel-type chelating preparation containing 1-hydroxyethylidene-1, 1-bisphosphonate

AIM: To test an aqueous gel containing 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) regarding its interactions with sodium hypochlorite, its calcium binding capacity, and its potential in preventing the formation of a smear layer when used in conjunction with rotary root canal preparation. METHODOLOGY: The experimental aqueous gel consisted of (w/v) 2% alginate, 3% aerosil, 10% Tween 80 and 18% HEBP. Interactions of gel components with hypochlorite were assessed using iodometric titration and monochromatic ultraviolet spectrometry. Two commercial paste-type chelators containing ethylenediaminetetraacetic acid (EDTA) and peroxide (RC-Prep and Glyde) served as controls. Calcium-binding capacities were measured in mixtures with a Ca2+ standard solution buffered at pH 10 using a calcium-selective measuring chain. Finally, root canals of 16 extracted single-rooted premolars per group were instrumented using ProFile instruments dipped in the experimental gel, RC-Prep, or nothing. Additionally, canals were rinsed with 10 mL of a 1% NaOCl solution during/after preparation. Smear scores in instrumented teeth were monitored using scanning electron microscopy. RESULTS: None of the experimental gel components showed short-term interactions with hypochlorite, whilst EDTA, peroxide, RC-Prep and Glyde immediately reduced the hypochlorite in solution. The experimental gel chelated 30 mg Ca2+ g-1, compared with 16 mg Ca2+ g-1 and 11 mg Ca2+ g-1 chelated by RC-Prep and Glyde respectively. Smear scores obtained with the experimental gel were significantly (P<0.05) lower than with RC-Prep in coronal and middle root thirds, whilst no differences were observed in apical root thirds. CONCLUSIONS: Under the conditions of this study, an HEBP gel appeared advantageous over currently available products.