Release of chlorpheniramine maleate from fatty acid ester matrix disks prepared by melt-extrusion.

Chlorpheniramine maleate was incorporated into disks consisting of glyceryl fatty acid esters, polyethylene glycol fatty acid esters or a combination of the two. A melt-extrusion process was used to prepare the matrix disks containing the drug. The release of the drug into distilled water, pH 1.2 buffer, and pH 7.5 buffer showed the expected square root of time dependence. An increase in the fatty acid ester hydrophilic-lipophilic balance (HLB) from 1 to 14 resulted in a 10-fold increase in the drug release rate from 0.25 +/- 0.01 to 25.84 +/- 1.29 mg cm-2 h-1/2. The maximum release rate was seen from the fatty acid ester with a melting point of 44 degrees C. The pH of the dissolution medium had a small impact on the rate of drug release, but the rate of agitation had no significant influence on the rate of drug release. By blending a fatty acid ester of a high melting point (64 degrees C) and a low HLB value of 2 with esters of lower melting points (33 to 50 degrees C) or higher HLB values (10 to 14), it was possible to modify the release from 10.0 +/- 0.70 to 21.5 +/- 0.57 mg cm-2 h-1/2.     

Colebroside A, a new diglucoside of fatty acid ester of glycerin from Clerodendrum colebrookianum

Colebroside A (1), a new diglucoside of fatty acid ester of glycerin, was isolated from the aerial parts of Clerodendrum colebrookianum Walp., along with nine known compounds (2-10). Their structures were elucidated by spectroscopic and chemical methods. Compounds 2, 3, 4, 5, 7, 8, 9 and 10 have been obtained from this plant for the first time.     

Ethanol-induced fatty acid ethyl ester formation in vivo and in vitro in rat lung

Fatty acid ethyl esters (FAEE) are the end products of a non-oxidative pathway for ethanol metabolism in a variety of human, rabbit, rat and murine tissues. Our objective was to determine the significance of this pathway in the metabolism of ethanol by the rat lung. In vitro, ¹⁴C-labeled ethyl oleate formation was assayed in the lung and compared with the pancreas, liver, heart and brain. Lipids were extracted with acetone, and ¹⁴C-labeled ethyl oleate was isolated and quantified by thin layer chromatography (TLC) and scintillation spectrometry. FAEE synthetic activity in the lungs (in vitro) was found to be intermediate among the organs examined. In vivo, male rats received 10% ethanol in their drinking water with or without daily i.p. injections of 4-methylpyrazole (1 mmol/kg body wt) for 15 days. Another group of male rats received 4 g/kg body wt ethanol as a 50% (v/v) solution by gavage every 12 h for 2 days. FAEE from the three organs with the highest in vitro activity for FAEE synthesis (pancreas, liver and lung) were extracted with acetone, isolated from normal lipids by TLC and separated by gas chromatography. The lung had lower FAEE-forming activity than the pancreas or the liver in the 15-day studies. However, in the 2-day study, the lung had higher activity than the liver but lower activity than the pancreas. Ethyl oleate, ethyl stearate and ethyl palmitate were the predominant FAEE formed in the intact organism. Ethanol-induced FAEE may play a role in the development of alcohol-related injuries to the lung.     

大鼠酸感受离子通道亚基2a表达质粒的构建及生物学特性考察

目的构建大鼠酸感受离子通道亚基2a(acid-sensingion channel subunit 2a,ASIC2a)的表达质粒并研究其组成的同聚体离子通道的生物学特性。方法使用分子生物学技术构建大鼠ASIC2a亚基表达质粒;通过体外转录技术,使编码ASIC2a亚基的cRNA在爪蟾卵母细胞内表达并在膜表面形成同聚体离子通道;使用双电极电压钳技术研究ASIC2a的生物学特性。结果在注射ASIC2a亚基cRNA的爪蟾卵母细胞上,降低胞外液pH值可诱导出内向电流。H+诱发的ASIC2a内向电流具有稳态失活成分可被氨氯吡咪可逆性阻断,其pH50为5.12。提高胞外Ca2+浓度可降低H+诱发的电流幅度,其IC50为11.98 mmol.L-1。当细胞外液中无Na+时,H+基本上不能诱发出内向电流;当同时去除细胞外液中Na+和K+时,H+可诱发出外向电流。结论成功构建ASIC2a表达质粒;ASIC2a除了对Na+通透外,对K+也有一定的通透性,胞外Ca2+抑制ASIC2a孔道的开放。     

Uptake and metabolism of the short-chain fatty acid butyrate, a critical review of the literature

Butyrate is a short-chain fatty acid (SCFA) formed by bacterial fermentation of fibre in the colon, and serves as an energy source for colonocytes. The action of butyrate as a histone deacetylase inhibitor (HDACi) has led to a number of clinical trials testing its effectiveness as a potential treatment for cancer. The biology of butyrate transport is therefore relevant to both its physiological and pharmacological benefits. This review of the literature was carried out to assess the evidence for both the uptake and metabolism of butyrate, in an attempt to determine possible mechanism (s) by which butyrate can act as an HDACi. It is noted that although uptake and metabolism are well characterised, there are still significant gaps in the knowledgebase around the intracellular handing of butyrate, where assumptions or dated evidence are relied upon.     

Absorption and metabolic characteristics of p-aminobenzoic acid and its isomer, m-aminobenzoic acid, from the rat small intestine.

Absorption and metabolic characteristics of p-aminobenzoic acid (PABA) and m-aminobenzoic acid (MABA) from the rat small intestine were examined by means of in situ recirculation and in vitro everted sac experiments. p-Aminobenzoic acid was extremely rapidly absorbed from the rat small intestine, whereas the absorption of MABA, the m-isomer of PABA, was comparably slower. This finding was partly explained by the result that PABA is more lipophilic than MABA. The metabolite percentage of PABA was considerably greater than that of MABA in mucosal fluid, tissue, and serosal fluid. On the other hand, a concentration-dependent and a directional difference in the transfer rate of these drugs were observed in everted and noneverted sacs of rat small intestine. Furthermore, mucosal uptake of PABA or MABA was inhibited by 1 mM 2,4-dinitrophenol, 10 mM sodium azide, and pretreatment with HgCl2 (10 mM). These results indicate that MABA, as well as PABA, is transported through the intestine by a carrier-mediated transport system, and that the molecular structure of these drugs is important for their absorption and metabolic characteristics.     

A new ester of fatty acid from a methanol extract of the whole plant of Amaranthus spinosus and its α-glucosidase inhibitory activity

Abstract

Context: Amaranthus spinosus Linn. (Amaranthaceae), commonly known as “spiny pigweed”, is used both in the Indian traditional system and in folk medicine to treat diabetes.

Objective: The present study evaluates the scientific basis of antidiabetic activity of chloroform fraction of methanol extract of A. spinosus and of an isolated constituent of A. spinosus.

Materials and methods: HPLC analysis was performed to determine the purity and the amount of the constituent present in the plant extract. The yeast α-glucosidase inhibition technique was used to determine the antidiabetic activity of A. spinosus. Acarbose was used as a standard. An appropriate therapeutic approach for preventing diabetes mellitus and obesity is to retard the absorption of glucose by inhibition of α-glucosidase.

Results: One novel fatty acid with strong α-glucosidase inhibitory activity – (14E, 18E, 22E, 26E) – methyl nonacosa-14, 18, 22, 26 tetraenoate [1] (IC₅₀ value 6.52?µM/mL) and β-sitosterol [2] were purified. Compound 1 was found to be more potent than the methanol extract. HPLC quantative analysis revealed that 0.15% of compound 1 and 0.06% of compound 2 were present in the plant extract.

Conclusion: This novel fatty acid can potentially be developed as a novel natural nutraceutical for the management of diabetes.     

Preparation of chondroitin sulfate-adipic acid dihydrazide-doxorubicin conjugate and its antitumour characteristics against LLC cells

Macromolecule–antitumour drug conjugates can reach tumour sites specifically via the enhanced permeability and retention (EPR) effect. It is desirable to release the drug efficiently from the conjugate at acidic pH in the tumour tissue or in the endosomes of cancer cells. In this study, we attempted to produce a carrier system with a labile chemical bond at acidic pH. Adipic acid dihydrazide (ADH)-chondroitin sulfate (CS) (termed CS-ACH) was synthesised by a two-step method, with the introduction of formyl groups followed by reductive amination using ADH. Doxorubicin (DOX) was conjugated to CS-ACH by simple mixing at acidic pH. The conjugate, designated CS-ACH-DOX, showed gradual drug release pH dependently at 37?°C; after incubation for seven days, more than 60% of DOX was released at pH 4, whereas less than 20% was released at pH 7. CS-ACH-DOX showed in vitro cytotoxicity against Lewis lung carcinoma (LLC) cells, which was less effective than that of DOX itself. However, CS-ACH-DOX inhibited tumour growth more than DOX in LLC tumour-bearing mice. These results suggested that CS-ACH-DOX might accumulate in tumours via the EPR effect and release DOX effectively at acidic pH. CS-ACH-DOX was considered to act as a drug delivery system with tumour targeting.     

Lack of toxicity or carcinogenicity of S-170, a sucrose fatty acid ester, in F344 rats.

A chronic combined toxicity and carcinogenicity study of S-170, a sucrose fatty acid ester, was performed in male and female F344 rats. S-170 was given ad libitum in the diet at levels of 0, 1.25, 2.5 or 5% to 10 rats/sex/group for 12 months to determine chronic toxicity and 0, 2.5 or 5% to 50 rats/sex/group for two years in the carcinogenicity study. Treatment with S-170 exerted no effect on survival in either sex. In the 12-month chronic toxicity study, no treatment-related effects on body weights, or hematological, blood biochemical, urinary and pathological parameters were demonstrated in any of the treated groups. In the carcinogenicity study, S-170 did not cause any dose-related significant increase in the incidences of tumors in any organs or tissues. Taken together, the results clearly demonstrate that S-170 has neither toxic nor carcinogenic activity in F344 rats under the conditions of the study. No observed adverse effect levels (NOAELs) calculated from the 12-month chronic toxicity and carcinogenicity study were 2.37 g/kg/day in males and 2.80 g/kg/day in females, and 2.12 g/kg/day in males and 2.42 g/kg/day in females, respectively.     

氯硝柳胺丙戊酸酯的体外化学稳定性及体内药代动力学初步研究

目的 对具有潜在的组蛋白去乙酰化酶和蛋白酪氨酸激酶双重抑制作用的抗肿瘤化合物氯硝柳胺丙戊酸酯进行体外水解动力学、血浆稳定性及体内药代动力学初步研究.方法 采用HPLC法研究体外氯硝柳胺丙戊酸酯在不同pH值的水溶液、血浆中的稳定性,以及体内研究大鼠静脉注射10 mg·kg-1氯硝柳胺丙戊酸酯后,测定不同时间点大鼠血浆中的氯硝柳胺丙戊酸酯及氯硝柳胺的浓度,对其血药浓度-时间采用BABP 3.0软件拟合,估算其药动学参数.结果 血浆中氯硝柳胺丙戊酸酯、氯硝柳胺在血浆中0.1-100.0 mg·L-1范围内线性关系良好(r=0.9999),回收率>80%和日内及日间精密度<10%;氯硝柳胺丙戊酸酯体外在酸性及中性水溶液中比较稳定,在碱性水溶液中易水解,在体外血浆中水解半衰期为8.5 min;氯硝柳胺丙戊酸酯以10 mg·kg-1静脉给药后,氯硝柳胺丙戊酸酯在大鼠体内的T12为(14.25±2.43) min,AUC0-t 为(280.6±39.7) min·mg·L-1,氯硝柳胺在大鼠体内的T12为(1.61±0.32) min,AUC0-t 为(57.1±20.4) min·mg·L-1.结论 氯硝柳胺丙戊酸酯在体外具有一定的稳定性,同时体内能够水解成氯硝柳胺和丙戊酸.     

Isolation of dealanylalahopcin, a new amino acid, and its biological activity

A new amino acid, dealanylalahopcin, was isolated from a culture filtrate of Streptomyces albulus subsp. ochragerus; it was also formed by the enzymatic hydrolysis of alahopcin using microbial alpha-amino acid ester hydrolase. The amino acid was obtained as colorless needles and its molecular formula is C6H10N2O5. It showed very weak antibacterial activity against Gram-positive and Gram-negative bacteria, and weak inhibitory activity against the collagen prolylhydroxylase.     

Inhibition of rat and human glutathione S-transferase isoenzymes by ethacrynic acid and its glutathione conjugate

Ethacrynic acid, a potent inhibitor of glutathione S-transferases (GST), has been shown to enhance the cytotoxicity of chlorambucil in drug resistant cell lines, but a definite mechanism has not been established. Both covalent binding to GST and reversible inhibition of GST have been reported. In the present study no irreversible inhibition was observed: for all rat GST tested, inactivation was complete within 15 sec at 0 degree, and dialysis of GST after incubation with ethacrynic acid gave complete recovery of enzyme activity for all isoenzymes tested. Moreover, the inhibition was competitive towards 1-chloro-2,4-dinitrobenzene and non-competitive towards glutathione for rat isoenzyme 1-1. Strong inhibition of both human and rat GST of the alpha-, mu- and pi-classes was obtained with ethacrynic acid, while conjugation of ethacrynic acid with glutathione did not abolish its inhibiting properties. For the alpha-, mu- and pi-class I50 values (microM) were 4.6-6.0, 0.3-1.9 and 3.3-4.8, respectively for ethacrynic acid, and 0.8-2.8, less than 0.1-1.2 and 11.0, respectively for its glutathione conjugate. Of all isoenzymes tested the human isoenzyme mu is most sensitive to the action of both ethacrynic acid and its glutathione conjugate.     

The physicochemical characteristics and bioavailability of indomethacin from beta-cyclodextrin, hydroxyethyl-beta-cyclodextrin, and hydroxypropyl-beta-cyclodextrin complexes

In an effort to improve the bioavailability of the insoluble drug indomethacin, three complexes were prepared with indomethacin and the soluble complexing agents β-, hydroxyethyl-β-, and hydroxypropyl-β-cyclodextrin. The indomethacin content was similar among the complexes (P≤0.05). To confirm complex formation, each complex was characterized by ultraviolet, infrared, nuclear-magnetic resonance, powder X-ray diffraction, and differential-scanning calorimetry techniques. Powder diffraction studies show the β-cyclodextrin complex was polycrystalline, and the hydroxyethyl- and hydroxypropyl-β-cyclodextrin complexes were amorphous. Phase-solubility analysis confirmed the formation of complexes and suggested the three complexes were bound similarly. Solubility studies show complexation increased indomethacin solubility, and the hydroxyethyl- and hydroxypropyl-β-cyclodextrin complexes were more soluble than the β-cyclodextrin complex in 0.1N hydrochloric acid and distilled water. Dosage forms were prepared by encapsulating the complexes without the addition of excipients. Dissolution studies show the encapsulated β- and hydroxyethyl-β-cyclodextrin complexes had superior dissolution when compared to the hydroxypropyl-β-cyclodextrin and Indocin^® (50 mg) capsules. Bioavailability studies were performed by administering the indomethacin complex or Indocin capsules to male-albino, New Zealand rabbits. Indomethacin plasma-time concentration data fit best to a compartment-independent model for all capsule formulations. Bioavailability comparisons by ANOVA show no significant difference (P≤0.10) in the peak-plasma time and peak concentration among the capsule formulations. The area-under-the-curve for the β-cyclodextrin complex capsules was found to be significantly higher (P≤0.10) than all other capsule formulations. In conclusion, the bioavailabilty of indomethacin was improved by complexation with only β-cyclodextrin. No correlations were found among the bioavailability, solubility, and dissolution results.     

Release of salicylic acid, diclofenac acid and diclofenac acid salts from isotropic and anisotropic nonionic surfactant systems across rat skin

Release of salicylic acid, diclofenac acid, diclofenac diethylamine and diclofenac sodium, from lyotropic structured systems, namely; neat and middle liquid crystalline phases, across mid-dorsal hairless rat skin into aqueous buffer were studied. Release results were compared with those from the isotropic systems. The donor systems composed of the surfactant polyoxyethylene (20) isohexadecyl ether, HCl buffer of pH 1 or distilled water and the specific drug. High performance liquid chromatography (HPLC) methods were used to monitor the transfer of the drugs across the skin barrier. Results indicated that the rate-determining step in the transport process was the release of the drug from the specified donor system. Further, apparent zero order release was demonstrated with all systems. Except for diclofenac sodium, drug fluxes decreased as the donor medium changed from isotropic to anisotropic. The decrease in fluxes was probably due to the added constrains on the movement of drug molecules. By changing the anisotropic donor medium from neat to middle phase, drug flux decreased in case of salicylic acid and diclofenac sodium. In the mean time, flux increased in case of the diethylamine salt and appeared nearly similar in case of diclofenac acid. Rates of drug transfer across the skin from the anisotropic donors seemed to be largely controlled by the entropy contribution to the transport process. The type and extent of drug-liquid crystal interactions probably influenced the latter.     

Release of ATP from rat urinary bladder mucosa: role of acid,vanilloids and stretch

Background and purpose:

ATP, released from urothelial cells, modulates afferent nerve firing from the urinary bladder. Here, we have characterized ATP release from the rat bladder mucosa in response to acid, capsaicin, electrical field stimulation (EFS) and stretch, using agonists and antagonists at transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs).

Experimental approach:

Rat mucosal strips (containing urothelium and lamina propria) in Perspex microbaths were superfused with Krebs solution. ATP was measured after exposure of matched strips to acid (pH 6.6–5.0), capsaicin (0.1–10 µM), EFS or stretch (150% of original length).

Key results:

Median basal ATP release was 3.46 nmol·g⁻¹. The mucosal strips responded to stimuli with potency order (median, IQR): acid (pH 5.6–6.0) 286 (103–555) > 10 µM capsaicin 188 (117–431) > 10 Hz EFS 63.0 (13.3–96.4) > stretch 24.4 (6.73–55.1) nmol ATP g⁻¹. ATP release in response to acid was pH dependent (P < 0.05). Responses to capsaicin did not desensitize nor were they concentration dependent. TRPV1 antagonist, capsazepine (10 µM) abolished capsaicin-evoked ATP release, and reduced acid-evoked (pH 6.5) release to 30% (P < 0.001). The ASIC channel antagonists gadolinium (0.1 mM) and amiloride (0.3 µM) reduced (P < 0.05) the acid-evoked (pH 6.5) release to 40 and 6.5% respectively. ASIC (ASIC1, ASIC2a, ASIC2b, ASIC3) and two TRPV1 gene products were detected in mucosal and detrusor extracts.

Conclusions and implications:

Capsaicin (at TRPV1) and acid (at both TRPV1 and ASIC) induce ATP release from the rat bladder mucosa. This ATP appears to be principally of urothelial origin. This study highlights the importance of ATP and acid as signalling molecules in modulating bladder function.     

Differential inhibition of hepatic, pancreatic, and plasma fatty acid ethyl ester synthase by tri-o-tolylphosphate in rats

Fatty acid conjugation of alcohols, catalyzed by fatty acid ethyl ester synthase (FAEES), results in the formation of lipophilic esters. Although the activity of FAEES is reported in almost all organs, including plasma, the interrelationship among various proteins expressing FAEES activity in different organs/tissues is not well understood. Earlier, we have reported an inhibition of FAEES activity in human hepatoma cells by tri-o-tolylphosphate (TOTP; serine esterase inhibitor). The present study was undertaken to further characterize the hepatic, plasma, and pancreatic FAEES in rats after ip injection of 10, 25, 50, or 100 mg/kg TOTP in corn oil or vehicle alone. After 18 h, animals were euthanized and FAEES activity in the plasma and postnuclear fractions of hepatic and pancreatic homogenates were assayed by measuring the ester formation following incubation with [1-(14)C]oleic acid and ethanol or methanol as substrates. Significant inhibition of FAEES activity was observed in hepatic postnuclear fraction. The esterase activity also showed a pattern similar to fatty acid ethyl and methyl ester synthesizing activity. A trend similar to hepatic synthesizing and hydrolyzing activities was also found in the plasma of TOTP-treated rats. However, no inhibition of synthetic activity toward formation of fatty acid ethyl or methyl esters or p-nitrophenyl acetate hydrolyzing activity was observed in the pancreas of rats after TOTP exposure. Our results also show that the protein expressing FAEES activity in the pancreas does not cross-react with antibodies to rat adipose tissue FAEES using Western blot analysis, which recognizes approximately 60- and approximately 84-kDa proteins in the liver and plasma, respectively. Furthermore, the inhibition in liver is at the functional level of enzyme as no change was observed between control and treated animals by immunohistochemistry. We conclude that fatty acid ethyl or methyl ester synthesizing enzyme(s) in the liver and plasma, which are inhibited by TOTP, are different from that present in the pancreas.