半合成头孢菌素抗生素的研究——Ⅱ.7-(7-或8-取代香豆素-3-甲酰胺基)头孢菌素的合成

本文报道了以7-或8-取代香豆素-3-羧酸为侧链酸,用酰氯法和Vilsmeier试剂法与7-ADCA,7-ACA和7-ACT缩合,合成了17个7-或8-取代香豆素-3-甲酰胺头孢菌素类衍生物,通过有机溶媒、葡聚糖凝胶(Sephadex LH-20)及离心薄层层析纯化精制,得到纯品。初步抑菌试验结果表明:化合物V₂,V₃,V₈,V₉,V₁₄和V₁₅对耐药性金黄色葡萄球菌有较强的作用。     

A comparison of HPLC and bioassay methods for plasma melanotan-II (MT-II) determination: Application to a pharmacokinetic study in rats

The pharmacokinetic profile of the melanotropic peptide, melanotan-II (MT-II), was determined in rats following a 0.3 mg kg^?1 intravenous dose. Regression analysis of the plasma MT-II concentrations determined using HPLC and bioassay methods indicated the existence of a significant linear correlation (r = 0.90, p < 0.001). The plasma concentration versus time plots determined using the two assay methods yielded biphasic disposition profiles that were essentially superimposable. The following pharmacokinetic parameters were assessed from plasma concentration versus time data using both methods: C_max, AUC, CL_s, t_1/2β, MRT V_dβ, and V_ss. Statistical comparison showed that the parameters measured by each method were not significantly different (at the 0.05 level) except for t_1/2β, MRT and V_ss. The presence of even one aberrant data point in the β-phase can significantly influence t_1/2β when only a few data points are available in the β-phase. Since MRT and V_ss were calculated from t_1/2β it is not surprising that these two parameters also differed between methods.     

Development of a novel method for predicting human volume of distribution at steady-state of basic drugs and comparative assessment with existing methods

The parameters characterizing tissue distribution refer to the tissue/plasma partition coefficients (Kp), which can be used to derive volume of distribution at steady-state (V_ss). The effort for predicting drug distribution in human has been further expanded to calculation methods using in vitro-based algorithms. The objective of the present study was to develop a novel prediction method to estimate human V_ss for moderate-to-strong bases. The predictive performance of the novel method was compared with other well established in vitro-based methods available in the literature. Relevant information collected from previous prediction studies of V_ss facilitated the development of the novel method. This was based on the calculation of V_ss from data on Kp, which were estimated by correlating the unbound tissue/plasma ratio in vivo (Kpu) with the unbound red blood cells partitioning (RBCu) determined in vitro. The comparative assessment of the novel correlation method with existing prediction methods of human V_ss was done using a literature dataset of 61 basic drugs (at least one pK_a ≥ 7). The five existing V_ss prediction methods published in the literature are comprised of four versions of tissue composition-based models along with the model of Lombardo using the principle of Oie-Tozer. The statistical analysis of the prediction performance indicated that the novel method demonstrated a greater degree of accuracy compared to all other published methods. The maximum percentage of predicted values that fall within a twofold-error range is 77% for the basic drugs tested. Overall, the present study describes the development and the assessment of the predictive performance of the novel prediction method of human V_ss based upon in vitro data, which appears to be superior based on the current dataset studied for basic drugs.     

凝胶色谱法同时测定香菇多糖制剂中多糖分子量和含量

目的 建立一种同时测定香菇多糖制剂中多糖分子量分布和含量的凝胶色谱方法.方法 采用TSKgel G5000PWxl、G4000PWxl、G3000PWxl （7.8 mm×30 cm,7μm）凝胶色谱柱串联,以右旋糖苷为对照品,0.1 mol· L-1乙酸钠-乙酸水溶液为流动相,通过示差检测器检测,柱温为60℃,流速为0.6 mL·min-1,进样量为100 μ上L.结果 Dextrans的分子量对数logM/W与峰保留时间在22～47 min内呈良好的线性关系（r=0.9990）;多糖进样量在20.12～201.20 μg范围内与峰面积呈良好的线性关系（r =0.9999）,加样回收率平均值为106.33％,RSD=1.55％（n=9）.结论 本方法简便、准确、重复性好,可用于香菇多糖注射制剂的质量控制.     

Modelling the loss of metabolic capacities of cultured hepatocytes: application to measurement of Michaelis-Menten kinetic parameters in in vitro systems

1. The loss of metabolic capacities during culture time constitutes a major limitation for the use of hepatocyte primary cultures in in vitro metabolism measurements. A new strategy is presented that permits one to calculate the Michaelis-Menten parameters V_max and K_m from extended experiments, by modelling V_max as a variable dependent on time using exponential or sigmoidal equations. 2. This method was tested with cortisol depletion in cultured rat hepatocytes. V_max and K_m were used to calculate intrinsic clearance, and comparisons were made with methods already described in the literature. Intrinsic clearances given by our method were scaled to in vivo hepatic clearances that were close to those reported in the literature. 3. Our method could quantify the V_max decrease with culture time from estimates of time parameters, t_1/2 or t₅₀. In our system, this V_max decrease was in agreement with P450 cytochrome inactivation rates published for the rat liver. 4. In conclusion, we propose a convenient, simple and useful general method for both Michaelis-Menten parameter estimation and modelling of variations in the metabolic capacities observed in in vitro systems. Such an approach should improve the usefulness of hepatocytes in primary cultures for long-term metabolism experiments.     

Stepwise determination of multicompartment disposition and absorption parameters from extravascular concentration-time data. Application to mesoridazine,flurbiprofen, flunarizine,labetalol, and diazepam

When disposition is monoexponential, extravascular concentrationtime (C, t) data yield both disposition and absorption parameters, the latter via the Wagner-Nelson method or deconvolution which are equivalent. Classically, when disposition is multiexponential, disposition parameters are obtained from intravenous administration and absorption data are obtained from extravascular C, tdata via the Loo-Riegelman or Exact Loo-Riegelman methods or via deconvolution. Thus, in multiexponential disposition one assumes no intrasubject variation in disposition, a hypothesis that has not been proven for most drugs. Based on the classical two and threecompartment open models with central compartment elimination, and using postabsorptive extravascular C, tdata only, we have developed four equations to estimate k₁₀ when disposition is biexponential and two other equations to estimate k₁₀ when disposition is triexponential. The other disposition rate constants are readily obtained without intravenous data. We have analyzed extravascular data of flurbiprofen (12 sets), mesoridazine (20 sets), flunarizine (5 sets), labetalol (9 sets), and diazepam (4 sets). In the case of diazepam intravenous C, tdata were also available for analysis. After disposition parameters had been estimated from the extravascular data the Exact Loo-Riegelman method with the Proost modification was applied to the absorptive extravascular data to obtain A_T/V_p as a function of time. These latter data for each subject and each drug studied were found to befitted by a function indicating either simple firstorder absorption, two consecutive firstorder processes, or zero order absorption. After absorption and disposition parameters had been estimated, for each set of extravascular data analyzed, a reconstruction trend line through the original C, tdata was made. The new methods allow testing of the hypothesis of constancy of disposition with any given drug. There is also a need for new methods of analysis since the majority of drugs have no marketed intravenous formulation, hence the classical methods cannot be applied.     

Isolation and characterization of a low molecular weight cadmium-binding protein from Syrian hamster lung

A low molecular weight cadmium-binding protein has been isolated from Syrian hamster lung. The protein elutes from a Sephadex G-75 column with $\text{V}{}_{\mathrm{<mtext>e</mtext>}}\text{V}{}_0$ (1.64 – 1.98) characteristic of metallothionein-like proteins and has an estimated molecular weight of 15,000. The Cd-binding protein does not appear to bind Cr³⁺, Ni²⁺, or Se⁴⁺. A low molecular weight Cd-binding protein in lungs of this species may be responsible for the prolonged retention of Cd in lung following pulmonary exposure of hamsters to CdCl₂.     

Miniaturized Forced Degradation of Therapeutic Proteins and ADCs by Agitation-Induced Aggregation Using Orbital Shaking of Microplates

Microplate-based formulation screening is a powerful approach to identify stabilizing excipients for therapeutic proteins while reducing material requirements. However, this approach is sometimes not representative of studies conducted in relevant container closures. The present study aimed to identify critical parameters for a microplate-based orbital shaking method to screen biotherapeutic formulations by agitation-induced aggregation. For this purpose, an in-depth methodological study was conducted using different shakers, microplates, and plate seals. Aggregation was monitored by size exclusion chromatography, turbidity, and backgrounded membrane imaging. Both shaker quality and liquid-seal contact had substantial impacts on aggregation during shaking and resulted in non-uniform sample treatment when parameters were not suitably selected. The well volume to fill volume ratio (V_well/V_fill) was identified as an useful parameter for achieving comparable aggregation levels between different microplate formats. An optimized method (2400 rpm [a_c 95 m/s²], V_fill 60-100 µL [V_well/V_fill 6-3.6], 24 h, RT, heat-sealed) allowed for uniform sample treatment independent of surface tension and good agreement with vial shaking results. This study provides valuable guidance for miniaturization of shaking stress studies in biopharmaceutical drug development, facilitating method transfer and comparability between laboratories.     

Investigation of lignin obtained by processing of Betula pendula with ionic liquids

Imidazolium-based ionic liquids bearing chloride, acetate, mesylate, and tosylate as anion were investigated for the treatment of birch (Betula pendula) bark to extract lignin. Although birch bark was previously extracted with methanol to remove low molecular weight organic compounds, various fatty acids and ferulic acid esters were analyzed in the lignin fraction using UPLC/ESI(?)-QTOFMS. GPC analysis shows differences in the molecular weight of the lignin samples obtained by treating bark with 1-butyl-3-methylimidazolium acetate and 1-ethyl-3-methylimidazolium tosylate. The use of 1-butyl-3-methylimidazolium chloride or 1-ethyl-3-methylimidazolium mesylate for the extraction of bark resulted in aromatic oligomers that are not fully soluble in the eluent used for GPC analysis. Therefore, the molecular weight as determined by GPC is only representative for the soluble part of these samples and not for the entire sample. Results obtained by investigation of the lignin samples extracted from bark using GPC and UPLC/ESI(?)-QTOFMS let us conclude that 1-butyl-3-methylimidazolium acetate is the most efficient ionic liquid among the solvents investigated for the extraction of lignin from bark. The high efficiency may be attributed to a high catalytic function of this ionic liquid in bound cleavage on the one hand and an active contribution of the acetate ion to trans-esterifications on the other hand.     

Evaluation of Prediction Accuracy for Volume of Distribution in Rat and Human Using In Vitro,In Vivo,PBPK and QSAR Methods

Volume of distribution at steady state (V_ss) is an important pharmacokinetic parameter of a drug candidate. In this study, V_ss prediction accuracy was evaluated by using: (1) seven methods for rat with 56 compounds, (2) four methods for human with 1276 compounds, and (3) four in vivo methods and three Kp (partition coefficient) scalar methods from scaling of three preclinical species with 125 compounds. The results showed that the global QSAR models outperformed the PBPK methods. Tissue fraction unbound (f_u,t) method with adipose and muscle also provided high V_ss prediction accuracy. Overall, the high performing methods for human V_ss prediction are the global QSAR models, Øie-Tozer and equivalency methods from scaling of preclinical species, as well as PBPK methods with Kp scalar from preclinical species. Certain input parameter ranges rendered PBPK models inaccurate due to mass balance issues. These were addressed using appropriate theoretical limit checks. Prediction accuracy of tissue Kp were also examined. The f_u,t method predicted Kp values more accurately than the PBPK methods for adipose, heart and muscle. All the methods overpredicted brain Kp and underpredicted liver Kp due to transporter effects. Successful V_ss prediction involves strategic integration of in silico, in vitro and in vivo approaches.     

测定低分子量透明质酸相对分子质量的3种方法比较

目的比较3种测定低分子量透明质酸(LMWHA)相对分子质量(Mr)方法。方法分别采用特性黏度法、高效凝胶渗透色谱(HPGPC)法及多角度激光散射仪结合凝胶渗透色谱(GPC/MALLS)法测定LMWHA的Mr,并对所得结果进行对比分析。结果对于Mr介于10 000~100 000范围内的LMWHA,特性黏度法测得的Mr值与GPC/MALLS法测得的Mr值基本相同,而HPGPC法由于所用标准Mr对照品结构上的差异,测得的Mr与其它两种方法测得的值差异较大。结论在一定Mr范围内,特性黏度法和GPC/MALLS法均可用于测定LMWHA的Mr。     

PROTEIN THERMOSTABILITY.

The amino acid compositions of more than 20 enzymes and protein from various closely related mesophilic and thermophilic micro-organisms (esp. Bacillus) have been used to calculate a variety of macroscopic parameters. These included the hydrophobic index (Hø), the ratio of polar to non-polar volumes (rho), the ratios of Arg/(Arg + Lys), and (Arg + Lys) or (Glx + Asx) to total amino acids, % H-bonding amino acids, %α-helix- or β-sheet-forming amino acids, the theoretical melting temperature (T_m^Ccalc), the total volume to total amino acid ratio (V_R), and the % non-polar residues (NPS). In contrast to previous similar comparisions with proteins from divergent sources, it was found that thermophilic vs mesophilic proteins from the same genus show correlations between thermostability and increased Hø, decreased rho, and increased Arg/(Arg + Lys), as well as increased α-index and β-index. Weaker correlations were seen for V_R, T_m^Calc, aliphatic index, and NPS, all derived from, or related to, Hø. No correlations existed for the other calculated parameters. These results are consistent with recent results of Argos et al. (1979) [Biochemistry 18, 5698–5703] on sequence analyses of glyceraldehyde-3-P dehydrogenases, where thermophilic proteins showed multiple amino acid replacements that caused increased internal hydrophobicity and increased external polarity. No trends were observed in any of the parameters calculated from amino acid compositions for crude cytoplasmic protein extracts from mesophilic vs thermophilic Bacilli.     

Systematic Study of Binding of μ-Conotoxins to the Sodium Channel NaV1.4

Voltage-gated sodium channels (Na_V) are fundamental components of the nervous system. Their dysfunction is implicated in a number of neurological disorders, such as chronic pain, making them potential targets for the treatment of such disorders. The prominence of the Na_V channels in the nervous system has been exploited by venomous animals for preying purposes, which have developed toxins that can block the Na_V channels, thereby disabling their function. Because of their potency, such toxins could provide drug leads for the treatment of neurological disorders associated with Na_V channels. However, most toxins lack selectivity for a given target Na_V channel, and improving their selectivity profile among the Na_V1 isoforms is essential for their development as drug leads. Computational methods will be very useful in the solution of such design problems, provided accurate models of the protein-ligand complex can be constructed. Using docking and molecular dynamics simulations, we have recently constructed a model for the Na_V1.4-µ-conotoxin-GIIIA complex and validated it with the ample mutational data available for this complex. Here, we use the validated Na_V1.4 model in a systematic study of binding other µ-conotoxins (PIIIA, KIIIA and BuIIIB) to Na_V1.4. The binding mode obtained for each complex is shown to be consistent with the available mutation data and binding constants. We compare the binding modes of PIIIA, KIIIA and BuIIIB to that of GIIIA and point out the similarities and differences among them. The detailed information about Na_V1.4-µ-conotoxin interactions provided here will be useful in the design of new Na_V channel blocking peptides.     

Effect of citreoviridin, a mycotoxin from Penicillium citreoviride, on kinetic constants of acetylcholinesterase and ATPase in synaptosomes and microsomes from rat brain

The subcutaneous injection of acute (6 mg/kg) and subacute doses of citreoviridin, a toxin from Penicillium citreoviride NRRL 2579, inhibited brain synaptosomal (Na⁺ + K⁺)-ATPase whereas in microsomes, both (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase activities are significantly stimulated in a dose-dependent manner. In vitro, both Mg²⁺ and (Na⁺ + K⁺)-ATPase activities are inhibited in synaptosomal as well as in microsomal fractions. Kinetic parameters obtained from double reciprocal plots indicate that under in vivo conditions citreoviridin increases V_max and K_m values of microsomal (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase, whereas in synaptosomes Mg²⁺-ATPase, V_max and K_m are unaltered but (Na⁺ + K⁺)-ATPase V_max is decreased and K_m increased. In vitro, both Mg²⁺ and (Na⁺ + K⁺)-ATPase K_m values are increased and V_max values are decreased in the synaptosomal as well as in the microsomal fractions. On the other hand, AChE V_max values are decreased and K_m values are increased in the synaptosomes under both in vivo and in vitro conditions. In microsomes, in vivo, the V_max values are decreased and K_m values are unaltered while in vitro both are unaltered. These results indicate that citreoviridin neurotoxicity may be related to the altered enzymatic activities.     

Development of a Preformulation Lipophilicity Screen Utilizing a C-18-Derivatized Polystyrene-Divinylbenzene High-Performance Liquid Chromatographic (HPLC) Column

Alkane/water partition coefficients have been predicted from the retention times of solutes using a C-18-derivatized polystyrene-divinylbenzene HPLC column (Act-I). Several classes of compounds, with molecular weights from 78 to 379 and partition coefficients ranging over several orders of magnitude, were included in the present study. A high correlation coefficient (0.953) was obtained from log-log plots of alkane/water partition coefficient versus capacity factor. A poor correlation was observed for octanol/water partition coefficients, presumably due to the hydrogen-bonding capability of octanol. The alkane/water correlation suggests that the system is devoid of significant specific solute-stationary phase interactions which are known to impart anomalous retention behavior to traditional reverse phase columns. Deviations of calculated alkane/water partition coefficients (and Hansch II_alkane coefficients) from observed values could not be explained in terms of solute (or substituent) polarizability, dipole moment, -para, or pK _HB values, further suggesting that specific interactions between the stationary phase and the solute are not significant. A molecular weight dependence that was independent of lipophilicity was observed. Thermodynamic and extrathermodynamic parameters of retention were obtained in order to investigate retention mechanisms for the Act-I column. The molecular weight dependence does not appear to be due to size exclusion or entropic expulsion of the solute from the stationary phase. Hansch II substituent coefficients calculated from retention times were found to be similar for benzene and steroid derivatives. Thus, the Act-I column may be utilized as a rapid lipophilicity screen for drug candidates of similar molecular weight.     

Interspecies Scaling: Predicting Volumes,Mean Residence Time and Elimination Half-life.* Some Suggestions

Extrapolation of animal data to assess pharmacokinetic parameters in man is an important tool in drug development. Clearance, volume of distribution and elimination half-life are the three most frequently extrapolated pharmacokinetic parameters. Extensive work has been done to improve the predictive performance of allometric scaling for clearance. In general there is good correlation between body weight and volume, hence volume in man can be predicted with reasonable accuracy from animal data. Besides the volume of distribution in the central compartment (V_c), two other volume terms, the volume of distribution by area (Vβ) and the volume of distribution at steady state (Vd_ss), are also extrapolated from animals to man. This report compares the predictive performance of allometric scaling for V_c, Vβ and Vd_ss in man from animal data. The relationship between elimination half-life (t 1/2) and body weight across species results in poor correlation, most probably because of the hybrid nature of this parameter. To predict half-life in man from animal data, an indirect method (CL = VK, where CL = clearance, V is volume and K is elimination rate constant) has been proposed. This report proposes another indirect method which uses the mean residence time (MRT). After establishing that MRT can be predicted across species, it was used to predict half-life using the equation MRT= 1.44 x t 1/2. The results of the study indicate that V_c is predicted more accurately than Vβ and Vd_ss in man. It should be emphasized that for first-time dosing in man, V_c is a more important pharmacokinetic parameter than Vβ or Vd_ss. Furthermore, MRT can be predicted reasonably well for man and can be used for prediction of half-life.     

Respiratory function measurements in clinical pharmacological studies including an assessment of the area under the MEFV curve as a new parameter in chronic bronchitic patients

Summary We have assessed the value of the area under the MEFV curve (AUC) as an index of respiratory function in chronic bronchitis and compared it with PFR, FEV₁, FVC, volume at 75% PFR (V₇₅), V₅₀, V₂₅, F₅₀ and F₇₅.The reproducibility of these parameters was tested in 10 normal subjects and 10 patients with chronic bronchitis. The FVC was the most reproducible while the coefficient of variation for the AUC was the same as for the other MEFV curve indices.The sensitivity (percentage change on bronchodilatation after intravenous aminophylline) of the above measurements was also tested in a further nine patients with chronic bronchitis. The AUC was much more sensitive to bronchodilatation than any of the other measurements.Therefore although the AUC was less reproducible than simple spirometric indices, it was more sensitive to bronchodilatation by a greater factor. This probably outweights its poor reproducibility and AUC would therefore seem to be a useful new index of bronchodilatation in chronic bronchitis.     

GC-MS法测定金银花中12种有机磷农药的残留量

目的：建立金银花中12种有机磷农药残留量的测定方法。方法：样品以丙酮超声提取,并采用凝胶渗透色谱（GPC）和氨基固相萃取柱（NH2-SPE）结合的方法进行净化,选用气相色谱与质谱串联技术（GC-MS）在DB．5MS毛细管柱上用程序升温技术分离,采用选择离子检测方式,以保留时间和特征离子进行目标成分的定性鉴别,外标法对样品进行测定。结果：12种有机磷农药在50-1000μg·L^-1范围内呈良好的线性关系,12种农药的方法定量限在3．6～7．2μg·k^-1之间,三个水平添加回收率在72．2l％～107．72％,RSD〈10％（n=9）。结论：本方法灵敏度高,重复性好,可用于全银花中12种有机磷农药残留的检测。     

Purification and characterization of Ts15, the first member of a new α-KTX subfamily from the venom of the Brazilian scorpion Tityus serrulatus

Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: α-, β-, γ- and κ-KTx. These peptides display varying selectivity and affinity for K_v channel subtypes. In this work, a novel toxin from the Tityus serrulatus venom was isolated, characterized and submitted to a wide electrophysiological screening on 5 different subtypes of Na_V channels (Na_V1.4; Na_V1.5; Na_V1.6; Na_V1.8 and DmNa_V1) and 12 different subtypes of K_V channels (K_V1.1 - K_V1.6; K_V2.1; K_V3.1; K_V4.2; K_V4.3; Shaker IR and ERG). This novel peptide, named Ts15, has 36 amino acids, is cross-linked by 3 disulfide bridges, has a molecular mass of 3956 Da and pI around 9. Electrophysiological experiments using patch clamp and the two-electrode voltage clamp techniques show that Ts15 preferentially blocks K_V1.2 and K_V1.3 channels with an IC₅₀ value of 196 ± 25 and 508 ± 67 nM, respectively. No effect on Na_V channels was observed, at all tested concentrations. Since Ts15 shows low amino acid identity with other known KTxs, it was considered a bona fide novel type of scorpion toxin. Ts15 is the unique member of the new α-Ktx21 subfamily and therefore was classified as α-Ktx21.1.     

Gel permeation chromatography of dextrans in parenteral solutions: calibration procedure development and method validation

We describe development and validation of a gel permeation chromatographic (GPC) method for dextrans in parenteral solutions. The GPC method was adopted from USP monographs on Dextran 40 and Dextran 70 raw materials. The method was optimized with a mobile phase flow rate of 1 mL/min and column temperature of 40 degrees C, to sharpen dextran and dextrose peaks. An easy-to-use, curve-fitting program capable of non-linear regression was developed in-house, using Microsoft Excel and its Solver add-in to successfully meet the GPC calibration requirements for dextrans and dextrose, i.e., the experimental molecular weights within 100+/-5% of the known molecular weights for dextrans and molecular weight of dextrose within 180+/-2 Da. The GPC method was validated in terms of its stability indicating nature, robustness (column temperature of 40+/-3 degrees C), accuracy (lack of effects of pH and concentration of dextrans or matrix components), and precision (repeatability and intermediate). Molecular weight distribution of dextrans were unchanged when the dextran containing test solutions were subjected to forced degradation using heat, light (daylight and UV light), extreme alkaline conditions or oxidative conditions. The method was capable of detecting changes in molecular weight distribution caused by degradation under extreme acidic conditions and heat, thereby confirming the stability indicating nature of the method. The concentration of Dextran 40 and Dextran 70 (75-125% of the nominal assay concentration), matrix components (108-111% of their nominal concentrations), and solution pH (pH 3-7 for Dextran 40 solutions and pH 4-7 for Dextran 70 solutions) did not affect the measured molecular weight distribution of Dextran 40 or Dextran 70. The method was precise with %R.S.D. of less than 1% for M (W) values of Dextran 40 or Dextran 70.