Ionization Constants and Distribution Coefficients of Phenothiazines and Calcium Channel Antagonists Determined by a pH-Metric Method and Correlation with Calculated Partition Coefficients

The pH–metric technique was used to determine the ionization constants and distribution coefficients of 10 phenothiazines and five ionizable calcium channel antagonists. Because the studied compounds were poorly water soluble and quite lipophilic with partition coefficients in the range of 3.5 to 5.5, organic cosolvents had to be added for the determination of the ionization constants to avoid precipitation of the free bases. The effect of the cosolvents dioxane and methanol on the extrapolation to pure water was compared. For both cosolvents a very good agreement with accessible published ionization constants was obtained, however the slope of the regression line was much smaller for dioxane, yielding more reliable estimates according to the standard deviation of the extrapolated values. Thus, dioxane might be preferable to methanol as a cosolvent for the determination of ionization constants of sparingly water soluble bases. Also the n‐octanol/water partition coefficients were determined and compared with published data and values calculated with the ClogP, ACD, and HINT programs. Although the obtained values were approximate in conformity with the published data, the calculated partition coefficients differed from the experimental ones considerably for the majority of the investigated compounds. Furthermore, the ion pair partitioning and the distribution coefficients at physiological pH 7.4 were determined. The pH‐dependent distribution profiles showed the strong influence of the ionization constants and of the distribution of the ion pairs on the overall distribution. This result strongly suggests that greater use should be made of measured distribution coefficients in quantitative structure–activity relationship studies. The potentiometric method is a convenient way to determine the distribution properties of drug molecules at pH values relevant for the biological system under investigation.     

Quantitative structure-activity relationships study of a series of imidazole derivatives as potential new antifungal drugs

The Quantitative Structure-Activity Relationships (QSAR) has been developed to relate antifungal activity against Candida albicans and Rhodotorula glutinis of new imidazole derivatives with their physico-chemical and structural properties. For 265 imidazole derivatives the most significant statistically equations has been obtained with correlation coefficients R=0.800 and R=0.820 in case of activity against Ca. and Rh.g., respectively. The overall antifungal activity has been described by means of size and bulkiness related parameters as well as polar and lipophilic interactions. The significance of lipophilicity in terms of n-octanol/water partition coefficient, ClogP, on antifungal potency against both fungi has been investigated. QSAR equations for different classes of antifungal activity have been obtained. With a very high probability level (92% and 96%) the weak or very weak antifungal potency against C.a. can be determined and thus the number of required experiments can be reduced.     

Comparative safety testing of genetically modified foods in a 90-day rat feeding study design allowing the distinction between primary and secondary effects of the new genetic event

This article discusses the wider experiences regarding the usefulness of the 90-day rat feeding study for the testing of whole foods from genetically modified (GM) plant based on data from a recent EU-project [Poulsen, M., Schr?der, M., Wilcks, A., Kroghsbo, S., Lindecrona, R.H., Miller, A., Frenzel, T., Danier, J., Rychlik, M., Shu, Q., Emami, K., Taylor, M., Gatehouse, A., Engel, K.-H., Knudsen, I., 2007a. Safety testing of GM-rice expressing PHA-E lectin using a new animal test design. Food Chem. Toxicol. 45, 364-377; Poulsen, M., Kroghsbo, S., Schr?der, M., Wilcks, A., Jacobsen, H., Miller, A., Frenzel, T., Danier, J., Rychlik, M., Shu, Q., Emami, K., Sudhakar, D., Gatehouse, A., Engel, K.-H., Knudsen, I., 2007b. A 90-day safety in Wistar rats fed genetically modified rice expressing snowdrop lectin Galanthus nivalis (GNA). Food Chem. Toxicol. 45, 350-363; Schr?der, M., Poulsen, M., Wilcks, A., Kroghsbo, S., Miller, A., Frenzel, T., Danier, J., Rychlik, M., Emami, K., Gatehouse, A., Shu, Q., Engel, K.-H., Knudsen, I., 2007. A 90-day safety study of genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in Wistar rats. Food Chem. Toxicol. 45, 339-349]. The overall objective of the project has been to develop and validate the scientific methodology necessary for assessing the safety of foods from genetically modified plants in accordance with the present EU regulation. The safety assessment in the project is combining the results of the 90-day rat feeding study on the GM food with and without spiking with the pure novel gene product, with the knowledge about the identity of the genetic change, the compositional data of the GM food, the results from in-vitro/ex-vivo studies as well as the results from the preceding 28-day toxicity study with the novel gene product, before the hazard characterisation is concluded. The results demonstrated the ability of the 90-day rat feeding study to detect the biological/toxicological effects of the new gene product in the GM food. The authors consider on this basis that the 90-day, rodent feeding study with one high dose level and a dietary design based upon compositional data on the GM food and toxicity data on the gene product is sensitive and specific enough to verify the presence/absence of the biological/nutritional/toxicological effects of the novel gene insert and further by the use of spiking able to separate potentially unintended effects of the novel gene product from other unintended effects at the level of intake defined in the test and within the remit of the test. Recommendations for further work necessary in the field are given.     

Physicochemical and cell-based approach for early screening of phospholipidosis-inducing potential

Some of the principal requisites of toxicity screening methods in drug discovery are their ease to perform and high throughput, as well as the possibility to predict the occurrence of clinical events. Phospholipidosis is one of the toxicities often induced by potential drugs. Several physicochemical methods for the prediction of phospholipidosis have been reported. The purpose of the present study was to examine the predictability of methods based on lipophilicity and charge parameters. We employed a test set of 33 compounds including 11 in-house compounds. The phospholipidosis-inducing potential (PLIP) of the test set compounds was determined by the fluorescence-labeled lipid accumulation assay using isolated rat hepatocytes. This assay was verified by transmission electron microscopy (EM). The usefulness of the ClogP - most basic pK(a) (pK(a) -MB) plot to the PLIP of compounds was examined. This plot was unable to predict the PLIP of zwitterions. In order to improve its predictability, the net charge of a given molecule (NC) was introduced instead of pK(a) - MB, since the NC corresponds directly to the ionization state of compounds in the organelles. Compounds with high ClogP (> 1) and high NC (1< or =NC< or =2) tended to be positive. This finding was also confirmed using 30 additional validation set compounds obtained from the literature. The ClogP - NC plot differentiated positive and negative compounds with more than 98% accuracy (62/63), indicating its usefulness in drug discovery.     

In silico assay for assessing phospholipidosis potential of small druglike molecules: training, validation, and refinement using several data sets

Phospholipidosis (PLD) is a lysosomal storage disorder induced by compounds, notably cationic amphiphilic drugs, which although reversible interferes with cellular phospholipids.The in silico method described utilizes the amphiphilic moment ΔΔG(AM) (kJ/mol) together with basic pK(a) values to assign PLD inducing potential to a compound. The new model was accurate and sensitive (85% and 82%, respectively) when compared to other data sets. Therefore, the parallel in vitro assay for PLD was discontinued. The data reinforce our view that the amphiphilic moment is far more informative for determining a compound's potential to induce PLD than the combined use of basic pK(a) and ClogP values.     

Non-invasive product temperature determination during primary drying using tunable diode laser absorption spectroscopy

The goal of this work was to demonstrate the application of Tunable Diode Laser Absorption Spectroscopy (TDLAS) as a non-invasive method to determine the average product temperature of the batch during primary drying. The TDLAS sensor continuously measures the water vapor concentration and the vapor flow velocity in the spool connecting the freeze-dryer chamber and condenser. Vapor concentration and velocity data were then used to determine the average sublimation rate (g/s) which was subsequently integrated to evaluate the amount of water removed from the product. Position dependent vial heat transfer coefficients (K(v)) were evaluated using the TDLAS sensor data for 20 mL vials during sublimation tests with pure water. TDLAS K(v) data showed good agreement to K(v) data obtained by the traditional gravimetric procedure. K(v) for edge vials was found to be about 20-30% higher than that of center vials. A weighted K(v) was then used to predict a representative average product temperature from TDLAS data in partial and full load freeze drying runs with 5%, 7.5%, or 10% (w/w) sucrose, mannitol, and glycine solutions. TDLAS product temperatures for all freeze-drying runs were within 1-2 degrees C of "center vial" steady state thermocouple data.     

Inhibition of syk activity and degranulation of human mast cells by flavonoids

To investigate the effect of flavonoids on the activation of p72(syk) (Syk) protein tyrosine kinase which plays a pivotal role in the high affinity IgE receptor-mediated degranulation of mast cells, we picked out 10 flavonoids, classified them into 4 series, and examined their effects on the activation of Syk and on the degranulation of human mast cells. Flavones and flavonols showed clear inhibition, whereas flavanones and isoflavones had either weak or no effect on Syk enzymatic activity induced by amino acid peptide corresponding to the activation loop domain and on IgE-dependent degranulation of human cultured mast cells (HCMC). On the basis of calculated logP (ClogP) values as a prediction of compound lipophilicity, some flavonoids were speculated to have low lipophilicity, the reason for poor cell permeability. A significant relationship was observed between the inhibition of Syk activity and HCMC degranulation attributable to flavonoids when the ClogP values of the compounds were taken into account (r(2)=0.89). These results suggested that the impairment of mast cell degranulation by several flavonoids classified into flavones and flavonols might be mediated via inhibition of the intracellular activation of Syk.     

Streptomyces hygroscopicus K2509利用离子交换废水制备培养基发酵生产井冈霉素A

本文筛选出一株对离子交换废水耐受性能较好的井冈霉素A发酵菌株Streptomyces hygroscopicus K2509。通过测定总糖、还原糖、发酵液电容、井冈霉素A产量等发酵参数，用摇瓶试验对比了S. hygroscopicus K2509和出发菌株S. hygroscopicus K18的差异，用15L罐试验验证了两菌株在离子交换废水配制培养基中的代谢特性。在摇瓶实验中，S. hygroscopicus K2509井冈霉素A产量低于出发菌株，但其表现出较好的离子交换废水耐受能力；在15L发酵罐实验中，S. hygroscopicus K18和K2509井冈霉素A产量分别为18.9和20.2g/L，产率分别为0.45和0.31g/L·h。新筛选菌株可在离子交换废水配制培养基中实现井冈霉素A正常发酵，可有效利用井冈霉素A提取工序所产生的废水，降低产品成本。     

Impaired inhibition of epileptiform activity by baclofen, but not by adenosine in the weaver hippocampus

The weaver defect results in a loss of baclofen- and adenosine-gated K+ conductance in the hippocampus of adult homozygous (wv/wv) mice. In addition, suppression of hippocampal epileptiform activity by baclofen is impaired (Jarolimek, W., B?urle, J., Misgeld, U., 1998. Pore mutation in a G protein-gated inwardly rectifying K+ channel subunit causes loss of K+ dependent inhibition in weaver hippocampus. Journal of Neuroscience 18, 4001-4007). We used wv/wv and wild-type (+/+) mice to determine whether K+ conductance increases are essential for the suppression of epileptiform activity by R-baclofen and adenosine in disinhibited hippocampal slices. In wv/wv mice R-baclofen was less potent by two orders of magnitude in reducing the frequency of spontaneous synchronous burst discharges than in +/+ mice. Endogenous adenosine and adenosine A1 receptor agonists differed only slightly in their efficacy to inhibit spontaneous synchronous burst discharges in wv/wv and +/+ mice. The findings on adenosine A1 receptors suggest that the varied efficacy of R-baclofen in wv/wv and +/+ mice may not be explained solely on the basis of a loss of ligand-gated K+ conductance. Therefore, we investigated the affinity of GABA(B) receptors for the antagonist CGP55845A in wv/wv and +/+ hippocampi. Schild plot analysis revealed a K(D) for the GABA(B) antagonist CGP55845A 10 fold higher in wv/wv than in +/+ mice. The data suggest that an alteration of GABA(B) receptors could contribute to the reduced efficacy of R-baclofen to suppress hippocampal epileptiform activity in weaver mice, while the suppression by adenosine remains largely unaffected.     

Intrinsic effects of AM4113, a putative neutral CB1 receptor selective antagonist, on open-field behaviors in rats

We examined open-field effects in rats of the cannabinoid 1 receptor (CB1R) agonist WIN55,212-2 (WIN; 3 mg/kg) and its interaction with the CB1R putative neutral antagonist AM4113 (0.3 to 3 mg/kg). Separate studies examined AM4113 alone (0.3 to 5.6 mg/kg). Unlike the CB1R antagonist rimonabant, in vitro (e.g., [Sink K.S., McLaughlin P.J., Wood J.A., Brown C., Fan P., Vemuri V.K., Pang Y., Olzewska T., Thakur G.A., Makriyannis A., Parker L.A., Salamone J.D. The novel cannabinoid CB(1) receptor neutral antagonist AM4113 suppresses food intake and food-reinforced behavior but does not induce signs of nausea in rats. Neuropsychopharmacology 2008a; 33: 946-955.; Sink K.S., Vemuri V.K., Olszewska T., Makriyannis A., Salamone J.D. Cannabinoid CB1 antagonists and dopamine antagonists produce different effects on a task involving response allocation and effort-related choice in food-seeking behavior. Psychopharmacology (Berl) 2008b; 196: 565-574.]) AM4113 produced no change in cAMP accumulation (neutral antagonism vis-a-vis inverse agonism). Recorded behaviors were: ambulation, rearing, circling, latency, scratching, grooming, defecation, urination and vocalization/squeaking. WIN reduced ambulation and rearing; AM4113 completely (ambulation) or partially (rearing) antagonized these behaviors. WIN alone resulted in circling and an increased latency to leave the start area; effects blocked by AM4113. AM4113 increased scratching and grooming, effects attenuated but not abolished by WIN. AM4113 alone tended to reduce ambulation and rearing and had no effect on latency or circling. AM4113 alone increased scratching and grooming. Effects on defecation, urination and vocalization were non-significant. The open-field effects of AM4113 are similar to those reported for rimonabant in rats. Yet, unlike the inverse agonists rimonabant and AM251, the putative neutral CB1R antagonist AM4113 did not produce signs of nausea in ferrets and rats ([Chambers A.P., Vemuri V.K., Peng Y., Wood J.T., Olszewska T., Pittman Q.J., Makriyannis A., Sharkey K.A. A neutral CB1 receptor antagonist reduces weight gain in rat. Am J Physiol Regul Integr Comp Physiol 2007; 293: R2185-2193.; Sink K.S., McLaughlin P.J., Wood J.A., Brown C., Fan P., Vemuri V.K., Pang Y., Olzewska T., Thakur G.A., Makriyannis A., Parker L.A., Salamone J.D. The novel cannabinoid CB(1) receptor neutral antagonist AM4113 suppresses food intake and food-reinforced behavior but does not induce signs of nausea in rats. Neuropsychopharmacology 2008a; 33: 946-955.; Sink K.S., Vemuri V.K., Olszewska T., Makriyannis A., Salamone J.D. Cannabinoid CB1 antagonists and dopamine antagonists produce different effects on a task involving response allocation and effort-related choice in food-seeking behavior. Psychopharmacology (Berl) 2008b; 196: 565-574.]).     

半夏水提取液逆转多药耐药细胞系K562/A02耐药性的研究

目的观察非细胞毒性浓度半夏水提取液对耐阿霉素的人白血病细胞系K562/A02多药耐药性的逆转作用,并探讨其逆转机制.方法采用MTT法测定半夏水提取液的细胞毒性及其对K562/A02细胞敏感性的影响,用流式细胞仪检测非细胞毒性浓度的半夏水提取液处理后K562/A02细胞膜表面糖蛋白P170表达的变化.结果半夏水提取液对K562/A02细胞有一定的细胞毒作用,非细胞毒性浓度半夏水提取液可显著降低阿霉素对K562/A02细胞的IC50,显著降低细胞膜糖蛋白P170的表达.结论半夏水提取液可部分逆转多药耐药细胞系K562/A02细胞对阿霉素的耐药性.     

7-Substituted 5-amino-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines as A2A adenosine receptor antagonists: a study on the importance of modifications at the side chain on the activity and solubility.

It was demonstrated in the early 1990s that adenosine exerts many physiological functions through the interaction with four different receptors, named A1, A2A, A2B, and A3. In the past few years, our group has been involved in the development of A2A antagonists, which led to the synthesis of SCH 58261 (1), the first potent and selective adenosine A2A antagonist, which has been widely used as a reference compound. In this paper, we present an extended series of pyrazolotriazolopyrimidines synthesized with the aim to investigate the influence of the substitutions on the pyrazole ring. The choice of the substituents was based on their capability to improve water solubility while retaining high affinity and selectivity at the human A2A adenosine receptor subtype. In this series, some structural characteristics that are important for activity, i.e., tricyclic structure, free amino group at 5-position, furan ring, and substituent at 7-position on the pyrazole moiety, have been maintained. We focused our attention on the nature of the phenyl ring substituent to improve water solubility. Following this strategy, we developed new compounds with good affinity and selectivity for A2A adenosine receptors, such as 8d (K(i) 0.12 nM; hA1/hA2A ratio = 1025; R(m) = 2.8), 8h (K(i) 0.22; hA1/hA2A ratio = 9818; R(m) = 3.4), 8i (K(i) 0.18 nM; hA1/hA2A ratio = 994; R(m) = 2.8), 8k (K(i) 0.13 nM; hA1/hA2A ratio = 4430; R(m) = 3.6), and 14b (K(i) 0.19 nM; hA1/hA2A ratio = 2273; R(m) = 2.7). All the new synthesized compounds have no significant interaction with either A2B or A3 receptor subtypes. This new series of compounds deeply enlightens some structural requirements to display high affinity and selectivity for the A2A adenosine receptor subtype, although our goal of identifying new compounds with increased water solubility was not completely achieved. On this basis, other strategies will be devised to improve this class of compounds with a profile that appears to be promising for treatment of neurodegenerative disorders, such as Parkinson's disease.     

Note: Prediction of Readmission in a Cohort of Alcoholics: A 2-Year Follow-Up

A cohort of 1, 192 first admissions to metropolitan public alcohol treatment programs was examined after 2 years. With the sample subdivided by number of readmissions, discriminant analysis based on demographic data, social history, and current drinking behavior provided only modest prediction of group membership (K =. 46), but may be sufficient to provide an alerting function for recidivism.