Comparison of metabolic pharmacokinetics of baicalin and baicalein in rats

Baicalin and baicalein, a flavone glucuronide and its aglycone, are bioactive constituents of Scutellariae Radix with various beneficial activities. We have characterized and compared the metabolic pharmacokinetics of baicalin and baicalein in rats. Baicalein was administered intravenously and orally to rats, and baicalin was orally administered. An HPLC method was used to determine the concentration of baicalein before and after hydrolysis using beta-glucuronidase/sulfatase. The pharmacokinetic parameters were calculated by using WINNONLIN. Unpaired Student's t-test was used for statistical comparison. The result showed that after intravenous administration of baicalein, 75.7% of the dose was circulating as its conjugated metabolites. After oral administration of baicalein, absorption of baicalein itself was negligible, whereas the glucuronides/sulfates of baicalein were predominant in the plasma. When compared with intravenous bolus administration with dose correction, the absolute absorption was 40%. When baicalin was administered orally, glucuronides and sulfates of baicalein were exclusively circulating in the plasma. The relative absorption for baicalin was 65% when compared with baicalein. Profound differences of serum profile and pharmacokinetics were observed between oral baicalein and baicalin. Baicalin demonstrated significantly later time to peak concentration (t(max)) and lower peak serum concentration (C(max)) of baicalein conjugated metabolites than baicalein, indicating baicalin was absorbed more slowly and to a lesser extent than baicalein.     

Antitumor effects of emodin on LS1034 human colon cancer cells in vitro and in vivo: Roles of apoptotic cell death and LS1034 tumor xenografts model

Emodin, an active natural anthraquinone derivative, is found in the roots and rhizomes of numerous Chinese medicinal herbs and exhibits anticancer effects on many types of human cancer cell lines. The aim of this study investigated that emodin induced apoptosis of human colon cancer cells (LS1034) in vitro and inhibited tumor nude mice xenografts bearing LS1034 in vivo. In in vitro study, emodin induced cell morphological changes, decreased the percentage of viability, induced G2/M phase arrest and increased ROS and Ca²⁺ productions as well as loss of mitochondrial membrane potential (ΔΨ_m) in LS1034 cells. Emodin-triggered apoptosis was also confirmed by DAPI staining and these effects are concentration-dependent. Western blot analysis indicated that the protein levels of cytochrome c, caspase-9 and the ratio of Bax/Bcl-2 were increased in LS1034 cells after emodin exposure. Emodin induced the productions of ROS and Ca²⁺ release, and altered anti- and pro-apoptotic proteins, leading to mitochondrial dysfunction and activations of caspase-9 and caspase-3 for causing cell apoptosis. In in vivo study, emodin effectively suppressed tumor growth in tumor nude mice xenografts bearing LS1034. Overall, the potent in vitro and in vivo antitumor activities of emodin suggest that it might be developed for treatment of colon cancer in the future.     

Flavonoid pharmacokinetics and tissue distribution after repeated dosing of the roots of Scutellaria baicalensis in rats

Scutellariae Radix (root of Scutellaria baicalensis, SR) contains numerous flavonoids such as baicalin, baicalein, and wogonin. This study investigated the pharmacokinetics and tissue distribution of flavonoids and their metabolites in rats after repeated dosing of a SR decoction. Sprague-Dawley rats were orally administered SR at 2 g/kg for seven doses. After the 7th dose, blood samples were withdrawn at specific times and organs, including the liver, kidney, lung, and brain, and collected. The concentrations of baicalein and wogonin in the serum and various tissues were assayed by HPLC before and after hydrolysis with glucuronidase and sulfatase. Baicalein and wogonin were not detected in the serum, and the molecules found were their glucuronides/sulfates. In tissues, the free forms of baicalein and wogonin appeared in the liver, kidney, and lung in addition to their glucuronides/sulfates. Baicalein was the major form in the lung, whereas baicalein glucuronides/sulfates were the major forms in the liver and kidney. Wogonin was the major form in the liver, kidney, lung, and traces of wogonin glucuronides/sulfates were detected in the kidney and liver. Neither baicalein and wogonin nor their glucuronides/sulfates were detected in the brain. In conclusion, the glucuronides/sulfates of baicalein and wogonin were exclusively present in the circulation, whereas their free forms appeared in the lung, liver, and kidney.     

Profound difference in pharmacokinetics between morin and its isomer quercetin in rats

Morin and quercetin are isomeric antioxidant flavonols widely distributed in plant foods and herbs. The pharmacokinetics of both flavonols at two doses were investigated and compared in rats. Parent forms and their glucuronides and sulfates in serum were determined by HPLC before and after enzymatic hydrolysis, respectively. After oral dosing of morin, both the parent form, morin, and its glucuronides and sulfates were present in the bloodstream. The conjugated metabolites predominated at the dose of 25 mg kg(-1), whereas the parent form was predominant at the dose of 50 mg kg(-1). Moreover, the AUC of morin parent form increased by a factor of 37 when the dose doubled, indicating that morin showed nonlinear pharmacokinetics. On the other hand, quercetin presented only as glucuronides and sulfates in the blood, indicating negligible bioavailability of quercetin, and the metabolites showed linear pharmacokinetics at the two doses studied. When considering the total AUC of parent form with conjugated metabolites, the extent of absorption of morin was 3 fold that of quercetin at the dose of 50 mg kg(-1). The results indicated that the difference in hydroxylation pattern on B-ring of flavonol markedly affected their fates in rats.     

Emodin,an 11β-hydroxysteroid dehydrogenase type 1 inhibitor,regulates adipocyte function in vitro and exerts anti-diabetic effect in ob/ob mice

Aim:

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a potent and selective inhibitor of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) with the ability to ameliorate metabolic disorders in diet-induced obese mice. In the present study, we investigated the effects of emodin on adipocyte function and the underlying mechanisms in vitro, and its anti-diabetic effects in ob/ob mice.

Methods:

3T3-L1 adipocytes were used for in vitro studies. 11β-HSD1A activity was evaluated with a scintillation proximity assay. The adipogenesis, glucose uptake, lipolysis and adiponectin secretion were investigated in 3T3-L1 adipocytes treated with emodin in the presence of active (corticosterone) or inactive glucocorticoid (11-dehydrocorticosterone). For in vivo studies, ob/ob mice were administered emodin (25 and 50 mg·kg⁻¹·d⁻¹, ip) for 26 d. On the last day of administration, the serum was collected and the mesenteric and perirenal fat were dissected for analyses.

Results:

Emodin inhibited the 11β-HSD1 activity in 3T3-L1 adipocytes in concentration- and time-dependent manners (the IC₅₀ values were 7.237 and 4.204 μmol/L, respectively, after 1 and 24 h treatment. In 3T3-L1 adipocytes, emodin (30 μmol/L) suppressed 11-dehydrocorticosterone-induced adipogenesis without affecting corticosterone-induced adipogenesis; emodin (3 μmol/L) reduced 11-dehydrocorticosterone-stimulated lipolysis, but had no effect on corticosterone-induced lipolysis. Moreover, emodin (3 μmol/L) partly reversed the impaired insulin-stimulated glucose uptake and adiponectin secretion induced by 11-dehydrocorticosterone but not those induced by corticosterone. In ob/ob mice, long-term emodin administration decreased 11β-HSD1 activity in mesenteric adipose tissues, lowered non-fasting and fasting blood glucose levels, and improved glucose tolerance.

Conclusion:

Emodin improves the inactive glucocorticoid-induced adipose tissue dysfunction by selective inhibition on 11β-HSD1 in adipocyte in vitro and improves glycemic control in ob/ob mice.     

The Metabolism of Vitavax by Rats and Rabbits

Abstract

1. Vitavax (carboxin) administered orally to rats and rabbits leads to the excretion of unchanged Vitavax both in the urine and faeces.

2. The major metabolites excreted in the urine were found to be glucuronides of phenols formed by hydroxylation of the parent compound. Vitavax sulphoxide did not appear to be a metabolite.

3. Administration of ¹⁴C-labelled Vitavax showed radioactivity in the salivary gland, liver, kidney and gastrointestinal tract; the compound did not undergo significant ring scission in vivo.     

Emodin inhibits human sperm functions by reducing sperm [Ca2+]i and tyrosine phosphorylation

Emodin, a bioactive anthraquinone widely used in Chinese traditional medicine, disrupts mouse testicular gene expression in vivo. In this study, we investigated the toxicity of emodin to human sperm in vitro. Different doses of emodin (25, 50, 100, 200 and 400 μM) were applied to ejaculated human sperm. The results indicated that 100, 200 and 400 μM emodin significantly inhibited the total motility, progressive motility and linear velocity of human sperm. In addition, sperm's ability to penetrate viscous medium together with progesterone induced capacitation and acrosome reaction was also adversely affected by emodin. In contrast, emodin did not affect sperm viability. Furthermore, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and tyrosine phosphorylation, which serve as key regulators of sperm function, were dose-dependently reduced by emodin (50–400 μM). These results suggest that emodin inhibits human sperm functions by reducing sperm [Ca²⁺]_i and suppressing tyrosine phosphorylation in vitro.     

Characterization of metabolites of worenine in rat biological samples using liquid chromatography–tandem mass spectrometry

The in vivo and in vitro metabolites of worenine in rat were identified or characterized using a specific and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. In vivo samples including rat urine, feces, and plasma samples were collected after ingestion of 25 mg/kg worenine to healthy rats. The in vivo and in vitro samples were cleaned up by a solid-phase extraction procedure (C18 cartridges) and a liquid–liquid extraction procedure, respectively. Then these pretreated samples were injected into a reversed-phase C18 column with mobile phase of methanol–ammonium acetate (2 mM, adjusted to pH 3.5 with formic acid) (60:40, v/v) and detected by an on-line MS/MS system. As a result, at least twenty-seven metabolites and the parent medicine were found in rat urine after ingestion of worenine. Seven metabolites and the parent medicine were identified or characterized in rat feces. Three metabolites and the parent medicine were detected in rat plasma. One metabolite was found in the rat intestinal flora incubation mixture, and three metabolites were characterized in the homogenized liver incubation mixture. The main phase I metabolism of worenine in rat was dehydrogenization, hydrogenation, hydroxylation, and demethylene reactions, and that of phase II was sulfation and glucuronidation.     

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics II: Studies with isolated perfused rat liver

1.?To elucidate the determining factors for elimination pathways of sulfate and glucuronide metabolites of xenobiotics, a single-pass perfusion of 4-methylumbelliferone (4MU) or p-nitrophenol (pNP) was performed with an isolated rat liver preparation.

2.?Without bovine serum albumin in the perfusion system, clearance calculated based on the unbound concentration in the liver clearly showed that the net efflux clearances (CL_eff) of sulfates from the sinusoidal membrane were much higher than those of glucuronides and that the biliary excretion clearances (CL_b) of glucuronides were approximately two times larger than those of sulfates.

3.?The ratios of CL_eff to CL_b were much higher for sulfates than those for glucuronides. The bile-oriented elimination of glucuronides or sinusoidal efflux-oriented elimination of sulfates was observed even using the perfusate including 3% bovine serum albumin, but the sinusoidal efflux of sulfates was extensively enhanced by bovine serum albumin in the perfusate. The mechanisms behind this stimulatory effect remain to be elucidated.

4.?For both compounds, CL_b of glucuronide was comparable with CL_b of sulfate, meaning that CL_b is not responsible for the biliary excretion of glucuronides at extensively higher rate than sulfates.

5.?Higher concentration of glucuronides in the liver, partly caused by much lower CL_eff of glucuronides than that of sulfates, is likely responsible for the bile-oriented excretion of glucuronides. The extensive sinusoidal efflux of sulfates, leading to the urine-oriented excretion, is attributed to the substantially higher CL_eff than CL_b.

6.?In conclusion, the sinusoidal efflux is an important factor for determining elimination pathways of both sulfates and glucuronides, although further studies are needed to clarify the mechanisms of the sinusoidal efflux.     

Emodin, a naturally occurring anthraquinone derivative, suppresses IgE-mediated anaphylactic reaction and mast cell activation

The high-affinity receptor for IgE (Fc?RI)-mediated activation of mast cells plays an important role in allergic diseases such as asthma, allergic rhinitis and atopic dermatitis. Emodin, a naturally occurring anthraquinone derivative in oriental herbal medicines, has several beneficial pharmacologic effects, such as anti-cancer and anti-diabetic activities. However, the anti-allergic effect of emodin has not yet been investigated. To assess the anti-allergic activity of emodin, in vivo passive anaphylaxis animal model and in vitro mouse bone marrow-derived mast cells were used to investigate the mechanism of its action on mast cells. Our results showed that emodin inhibited degranulation, generation of eicosanoids (prostaglandin D₂ and leukotriene C₄), and secretion of cytokines (TNF-α and IL-6) in a dose-dependent manner in IgE/Ag-stimulated mast cells. Biochemical analysis of the Fc?RI-mediated signaling pathways demonstrated that emodin inhibited the phosphorylation of Syk and multiple downstream signaling processes including mobilization of intracellular Ca²⁺ and activation of the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and NF-κB pathways. When administered orally, emodin attenuated the mast cell-dependent passive anaphylactic reaction in IgE-sensitized mice. Thus, emodin inhibits mast cell activation and thereby the anaphylactic reaction through suppression of the receptor-proximal Syk-dependent signaling pathways. Therefore, emodin might provide a basis for development of a novel anti-allergic drug.     

Intestinal phase-II metabolism of quercetin in HT29 cells, 3D human intestinal tissues and in healthy volunteers: a qualitative comparison using LC-IMS-MS and LC-HRMS

1. Flavonoids are a large class of dietary molecules, among which quercetin is the most ubiquitous, which undergo an extensive intestinal phase-II metabolism. We compared the in vivo metabolism of quercetin in healthy volunteers with two in vitro models, HT29 cells and 3?D human intestinal tissues. Supernatants of the in vitro experiments and the human intestinal fluids (HIF) were analyzed by LC-IMS-MS and LC-HRMS in a qualitative way.

2. Quercetin glucuronides, sulfates and their methyl conjugates were detected in all three systems. The metabolic profiles were found to be different, both in terms of the metabolites produced and their relative proportions. In particular, quercetin sulfates were almost absent in supernatants from HT29 cells incubations while they were a major metabolite in HIF and also found in 3?D intestinal tissues incubations.

3. IMS provided structural information as well as a third dimension of characterization, while HRMS brought increased sensitivity and MS/MS confirmation. HT29 cells are a useful tool to generate phase-II metabolites but do not represent the in vivo situation. 3?D intestinal tissues appear as a more relevant tool to study the intestinal phase-II metabolism of flavonoids.

     

The rate-limiting step in the biliary elimination of some substrates of uridine diphosphate glucuronyltransferase in the rat

Phenolphthalein, 4-methylumbelliferone and 8-hydroxychinoline mutually inhibit each others enzymatic conjugation by microsomal UDP glucuronyltransferase (EC 2.4.1.17; acceptor unspecific) in vitro. After intravenous injection these UDP glucuronyltransferase substrates are excreted in the rat in bile as β-d-glucuronides. When these glucuronides were injected i.v. they were excreted partially in the bile and also in the urine. Ligation of the kidneys caused an increased biliary excretion of the i.v. injected glucuronides. To determine if these UDP glucuronyltransferase substrates also inhibited each others glucuronidation in vivo, two compounds were injected i.v. simultaneously into rats and the mutual effects on the biliary excretion of these compounds were studied. Phenolphthalein inhibited the biliary excretion of 4-methylumbelliferone in the form of its glucuronide conjugate in bile. This inhibition was not due to substrate competition for UDP glucuronyltransferase but to inhibition of excretion of the formed glucuronides from the liver cell into bile. From further experiments in which the mutual effects of the i.v. injected glucuronides on each others biliary excretion were studied it could be concluded that the rate-limiting step in the biliary elimination of phenolphthalein, 4-methylumbelliferone and 8-hydroxychinoline as glucuronides in the rat was the excretion of the products of UDP glucuronyltransferase activity in vivo into the lumen of the bile canaliculus and not conversion by UDP glucuronyltransferase.     

Emodin protects against high-fat diet-induced obesity via regulation of AMP-activated protein kinase pathways in white adipose tissue

Emodin is an active herbal component traditionally used in China for treating a variety of diseases. The aim of this study was to examine the effect of emodin on the reducing lipid accumulation in white adipose tissue of high-fat diet-fed rats, and on the regulation of the expression of the genes involved in lipid metabolism to elucidate the mechanisms. After being fed a high-fat diet for two weeks, rats were dosed orally with emodin (20, 40, 80 mg/kg/day) or pioglitazone (20 mg/kg/day), once daily for eight weeks. Changes in body weight, feeding pattern, serum lipids, coronary artery risk index, and atherogenic index were investigated. Subcutaneous white adipose tissues were isolated for pathology histology and Western blot analyses. Changes of triglyceride accumulation in differentiated 3 T3-L1 adipocytes were also investigated. Emodin exhibited a significant concentration-dependent decrease in the intracellular accumulation of triglyceride in 3 T3-L1 adipocytes. Emodin (80 mg/kg/day) displayed similar characteristics to pioglitazone (20 mg/kg/day) in reducing body weight gain and plasma lipid levels as well as the coronary artery risk and atherogenic indices of high-fat diet-fed rats. Emodin also caused dose related reductions in epididymal white adipose tissue sizes in high-fat diet-fed rats. Emodin and pioglitazone enhanced the phosphorylation of AMP-activated protein kinase and its primary downstream targeting enzyme, acetyl-CoA carboxylase, upregulated gene expression of carnitine palmitoyl transferase 1, and downregulated sterol regulatory element binding protein 1 and fatty acid synthase protein levels in the epididymal white adipose tissue of high-fat diet-fed rats. Our findings suggest that emodin could attenuate lipid accumulation in white adipose tissue through AMP-activated protein kinase activation.     

The anthraquinone derivative emodin attenuates methamphetamine-induced hyperlocomotion and startle response in rats

Abnormal signaling mediated by epidermal growth factor (EGF) or its receptor (ErbB) is implicated in the neuropathology of schizophrenia. Previously, we found that the anthraquinone derivative emodin (3-methyl-1,6,8-trihydroxyanthraquinone) inhibits ErbB1 signaling and ameliorates behavioral deficits of the schizophrenia animal model established by EGF challenge. In the present study, we assessed acute and subchronic effects of its administration on methamphetamine-triggered behavioral hyperactivation in rats. Prior subchronic administration of emodin (50 mg/kg/day, 5 days, p.o.) suppressed both higher acoustic startle responses and hyperlocomotion induced by acute methamphetamine challenge. In parallel, emodin also attenuated methamphetamine-induced increases in dopamine and its metabolites and decreases in serotonin and its metabolites. Emodin administered alone also had an effect on stereotypic movement but no influence on horizontal or vertical locomotor activity. In contrast to pre-treatment, post-treatment with emodin had no effect on behavioral sensitization to methamphetamine. Administration of emodin in parallel to or following repeated methamphetamine challenge failed to affect hyperlocomotion induced by methamphetamine re-challenges. These findings suggest that emodin has unique pharmacological activity, which interferes with acute methamphetamine signaling and behavior.     

Effect of estrogen-progestin combinations on the hepatic microsomal metabolism of mestranol

Three combinations of steroid contraceptive drugs (mestranol plus lynestrenol. norethindrone or norethynodrel) were given orally at effective antifertility doses for 30 days to rats, mice and guinea pigs. Eighteen hr after the last treatment, the animals were sacrificed for preparation of liver microsomal enzymes and the evaluation of mestranol metabolism in vitro.The results obtained indicate that these three animal species convert mestranol into ethynylestradiol. a hormonally active agent, which is further metabolized into more polar metabolites.A prior administration of contraceptive agents increases in rats and mice the disappearance of mestranol and the metabolism of ethynylestradiol in vitro. In guinea pigs the effect was much less marked.The results are discussed considering that the estrogen activity of mestranol in vivo depends on the availability of ethynylestradiol for the estrogen receptors.     

Isolation, identification and antiviral activities of metabolites of calycosin-7-O-β-D-glucopyranoside

In vivo and in vitro metabolites of calycosin-7-O-β-d-glucopyranoside in rats were identified using a specific and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MSⁿ) method. The parent compound and twelve metabolites were found in rat urine after oral administration of calycosin-7-O-β-d-glucopyranoside. The parent compound and six metabolites were detected in rat plasma. In heart, liver, spleen, lung and kidney samples, respectively, six, eight, seven, nine and nine metabolites were identified, in addition to the parent compound. Three metabolites, but no trace of parent drug, were found in the rat intestinal flora incubation mixture and feces, which demonstrated cleavage of the glycosidic bond of the parent compound in intestines. The main phase I metabolic pathways of calycosin-7-O-β-d-glucopyranoside in rats were deglycosylation, dehydroxylation and demethylation reactions; phase II metabolism included sulfation, methylation, glucuronidation and glycosylation (probably). Furthermore, two metabolites commonly found in rat urine, plasma and tissues were isolated from feces and characterized by NMR. The antiviral activities of the metabolite calycosin against coxsackie virus B₃ (CVB₃) and human immunodeficiency virus (HIV) were remarkably stronger than those of calycosin-7-O-β-d-glucopyranoside.     

Elucidation of Arctigenin Pharmacokinetics After Intravenous and Oral Administrations in Rats: Integration of In Vitro and In Vivo Findings via Semi-mechanistic Pharmacokinetic Modeling

Although arctigenin (AR) has attracted substantial research interests due to its promising and diverse therapeutic effects, studies regarding its biotransformation were limited. The current study aims to provide information regarding the pharmacokinetic properties of AR via various in vitro and in vivo experiments as well as semi-mechanistic pharmacokinetic modeling. Our in vitro rat microsome incubation studies revealed that glucuronidation was the main intestinal and liver metabolic pathway of AR, which occurred with V_max, K_m, and Cl_int of 47.5 ± 3.4 nmol/min/mg, 204 ± 22 μM, and 233 ± 9 μl/min/mg with intestinal microsomes and 2.92 ± 0.07 nmol/min/mg, 22.7 ± 1.2 μM, and 129 ± 4 μl/min/mg with liver microsomes, respectively. In addition, demethylation and hydrolysis of AR occurred with liver microsomes but not with intestinal microsomes. In vitro incubation of AR and its metabolites in intestinal content demonstrated that glucuronides of AR excreted in bile could be further hydrolyzed back to the parent compound, suggesting its potential enterohepatic circulation. Furthermore, rapid formation followed by fast elimination of arctigenic acid (AA) and arctigenin-4′-O-glucuronide (AG) was observed after both intravenous (IV) and oral administrations of AR in rats. Linear pharmacokinetics was observed at three different doses for AR, AA, and AG after IV administration of AR (0.48–2.4 mg/kg, r² > 0.99). Finally, an integrated semi-mechanistic pharmacokinetic model using in vitro enzyme kinetic and in vivo pharmacokinetic parameters was successfully developed to describe plasma concentrations of AR, AA, and AG after both IV and oral administration of AR at all tested doses.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-014-9664-x) contains supplementary material, which is available to authorized users.KEY WORDS: arctigenic acid, arctigenin, arctigenin-4′-O-glucuronide, pharmacokinetics, semi-mechanistic pharmacokinetic modeling     

Sodium sulphomethyl derivatives of polymyxins

Variations in the treatment of polymyxin B and polymyxin E (colistin) with formaldehyde and sodium bisulphite produce sulphomethyl derivatives which differ quantitatively in acute toxicity and in antibacterial activities in vitro and in vivo. The acute intravenous LD50 values of some sixty samples of these derivatives range from six- to more than eighty-fold those of the parent antibiotics; the in vitro antibacterial activities range from 2 to 12% and the in vivo activities from 20 to 50% of those of the parent antibiotics, with the most toxic derivatives showing the highest activities. When short and prolonged incubation methods are used, assays of the derivatives in solutions of different ages and of blood collected from man and dog after intramuscular injection, show that the antibacterial activities of these sulphomethyl derivatives depend on reversion to the unsubstituted form, and that the differences in the activities are due to variations in stability. These conclusions are supported by comparison of these sulphomethyl derivatives with stable acetyl derivatives. The lower in vivo activity is due, at least partly, to the high renal excretion of the substituted form. Electrophoresis shows that the derivatives are composite, the components corresponding to mono- to pentasulphomethyl polymyxin. Pain at the injection site is the most troublesome side-effect of polymyxin therapy, and this is avoided with these derivatives. In rats injected with quantities some twenty-times the usual human dose, the derivatives cause transitory decrease in urinary output and transitory proteinuria. After intramuscular injection of these derivatives into dogs, no antibiotic is detectable in the cerebrospinal fluid and concentrations present in the bile are not significantly different from those after injection of the parent antibiotic. When injected intracisternally into these animals, derivatives are less toxic than the parent compounds. These studies show that acute intravenous toxicity is a useful index of therapeutic efficiency and that derivatives with intravenous LD50 values of about 100 mg/kg are the most satisfactory ones. Because activity depends on reversion to the parent antibiotic, the use of these derivatives for topical application is contraindicated.     

An intravenous clarithromycin lipid emulsion with a high drug loading,H-bonding and a hydrogen-bonded ion pair complex exhibiting excellent antibacterial activity

The aim of this study was to develop an intravenous clarithromycin lipid emulsion (CLE) with good stability and excellent antibacterial activity. The CLE was prepared by the thin-film dispersed homogenization method. The interaction between clarithromycin (CLA) and cholesteryl hemisuccinate (CHEMS) was confirmed by DSC, FT-IR and ¹H NMR analysis. The interfacial drug loading, thermal sterilization, freeze–thaw stability, and in vitro and in vivo antibacterial activity were investigated systematically. DSC, FT-IR and ¹H NMR spectra showed that CHEMS (CLA: CHEMS, M ratio 1:2) could interact with CLA through H-bonding and a hydrogen-bonded ion pair. The CHEMS was found necessary to maintain the stability of CLE. Ultracentrifugation showed that almost 88% CLA could be loaded into the interfacial layer. The optimized CLE formulation could withstand autoclaving at 121 °C for 10 min and remain stable after three freeze–thaw cycles. The in vitro susceptibility test revealed that the CLA–CHEMS ion-pair and CLE have similar activity to the parent drug against many different bacterial strains. The in vivo antibacterial activity showed that the ED₅₀ of intravenous CLE was markedly lower than that of CLA solution administrated orally. CLE exhibited pronounced antibacterial activity and might be a candidate for a new nanocarrier for CLA with potential advantages over the current commercial formulation.     

Emodin inhibits growth and induces apoptosis in an orthotopic hepatocellular carcinoma model by blocking activation of STAT3

BACKGROUND AND PURPOSE

Aberrant activation of STAT3 is frequently encountered and promotes proliferation, survival, metastasis and angiogenesis in hepatocellular carcinoma (HCC). Here, we have investigated whether emodin mediates its effect through interference with the STAT3 activation pathway in HCC.

EXPERIMENTAL APPROACH

The effect of emodin on STAT3 activation, associated protein kinases and apoptosis was investigated using various HCC cell lines. Additionally, we also used a predictive tumour technology to analyse the effects of emodin. The in vivo effects of emodin were assessed in an orthotopic mouse model of HCC.

KEY RESULTS

Emodin suppressed STAT3 activation in a dose- and time-dependent manner in HCC cells, mediated by the modulation of activation of upstream kinases c-Src, JAK1 and JAK2. Vanadate treatment reversed emodin-induced down-regulation of STAT3, suggesting the involvement of a tyrosine phosphatase and emodin induced the expression of the tyrosine phosphatase SHP-1 that correlated with the down-regulation of constitutive STAT3 activation. Interestingly, silencing of the SHP-1 gene by siRNA abolished the ability of emodin to inhibit STAT3 activation. Finally, when administered i.p., emodin inhibited the growth of human HCC orthotopic tumours in male athymic nu/nu mice and STAT3 activation in tumour tissues.

CONCLUSIONS AND IMPLICATIONS

Emodin mediated its effects predominantly through inhibition of the STAT3 signalling cascade and thus has a particular potential for the treatment of cancers expressing constitutively activated STAT3.