Evaluation of diuretic efficacy and antiurolithiatic potential of ethanolic leaf extract of Annona squamosa Linn. in experimental animal models.

OBJECTIVE: The study is to investigate the diuretic and antiurolithiatic activities of ethanolic leaf extract of Annona squamosa Linn. in experimental animals. MATERIALS AND METHODS: For both studies, Wistar albino rats and two doses of extract (250 and 500 mg/kg) were used. Diuretic activity was evaluated by Lipschitz model. Urine volume and urine pH were noted, the concentration of sodium and potassium was estimated by flame photometry, and diuretic index, natriuretic index, and Lipschitz values were calculated from the results. Furosemide was used as a positive control. Ethylene glycol-induced urolithiasis model was used for antiurolithiatic study. Urine volume, urine pH, body weight, and biochemical parameters such as calcium, urea, uric acid, and creatine both from serum and urine were estimated. Antioxidant parameters and histopathological analysis of the kidney were evaluated. Cystone was used as a positive control in this study. Results were expressed as mean ± standard error of mean. Statistical analysis was carried out using one-way analysis of variance, followed by Dunnett's multiple comparison tests. RESULTS: In both diuretic and antiurolithiatic studies, both doses of the extract showed efficacy, and the dose of 500 mg/kg has shown a significant effect compared to positive control and negative control. CONCLUSION: The dose of 500 mg/kg showed a promising diuretic and antiurolithiatic activity.

In vitro metabolism and stability of the actinide chelating agent 3,4,3-LI(1,2-HOPO).

The hydroxypyridinonate ligand 3,4,3-LI(1,2-HOPO) is currently under development for radionuclide chelation therapy. The preclinical characterization of this highly promising ligand comprised the evaluation of its in vitro properties, including microsomal, plasma, and gastrointestinal fluid stability, cytochrome P450 inhibition, plasma protein binding, and intestinal absorption using the Caco-2 cell line. When mixed with active human liver microsomes, no loss of parent compound was observed after 60 min, indicating compound stability in the presence of liver microsomal P450. At the tested concentrations, 3,4,3-LI(1,2-HOPO) did not significantly influence the activities of any of the cytochromal isoforms screened. Thus, 3,4,3-LI(1,2-HOPO) is unlikely to cause drug-drug interactions by inhibiting the metabolic clearance of coadministered drugs metabolized by these enzymes. Plasma protein-binding assays revealed that the compound is protein-bound in dogs and less extensively in rats and humans. In the plasma stability study, the compound was stable after 1 h at 37°C in mouse, rat, dog, and human plasma samples. Finally, a bidirectional permeability assay demonstrated that 3,4,3-LI(1,2-HOPO) is not permeable across the Caco-2 monolayer, highlighting the need to further evaluate the effects of various compounds with known permeability enhancement properties on the permeability of the ligand in future studies.

A systematic screen of FDA-approved drugs for inhibitors of biological threat agents.

BACKGROUND: The rapid development of effective medical countermeasures against potential biological threat agents is vital. Repurposing existing drugs that may have unanticipated activities as potential countermeasures is one way to meet this important goal, since currently approved drugs already have well-established safety and pharmacokinetic profiles in patients, as well as manufacturing and distribution networks. Therefore, approved drugs could rapidly be made available for a new indication in an emergency. METHODOLOGY/PRINCIPAL FINDINGS: A large systematic effort to determine whether existing drugs can be used against high containment bacterial and viral pathogens is described. We assembled and screened 1012 FDA-approved drugs for off-label broad-spectrum efficacy against Bacillus anthracis; Francisella tularensis; Coxiella burnetii; and Ebola, Marburg, and Lassa fever viruses using in vitro cell culture assays. We found a variety of hits against two or more of these biological threat pathogens, which were validated in secondary assays. As expected, antibiotic compounds were highly active against bacterial agents, but we did not identify any non-antibiotic compounds with broad-spectrum antibacterial activity. Lomefloxacin and erythromycin were found to be the most potent compounds in vivo protecting mice against Bacillus anthracis challenge. While multiple virus-specific inhibitors were identified, the most noteworthy antiviral compound identified was chloroquine, which disrupted entry and replication of two or more viruses in vitro and protected mice against Ebola virus challenge in vivo. CONCLUSIONS/SIGNIFICANCE: The feasibility of repurposing existing drugs to face novel threats is demonstrated and this represents the first effort to apply this approach to high containment bacteria and viruses.

Lead optimization of antimalarial propafenone analogues.

Previously reported studies identified analogues of propafenone that had potent antimalarial activity, reduced cardiac ion channel activity, and properties that suggested the potential for clinical development for malaria. Careful examination of the bioavailability, pharmacokinetics, toxicology, and efficacy of this series of compounds using rodent models revealed orally bioavailable compounds that are nontoxic and suppress parasitemia in vivo. Although these compounds possess potential for further preclinical development, they also carry some significant challenges.

Development of a new generation of 4-aminoquinoline antimalarial compounds using predictive pharmacokinetic and toxicology models.

Among the known antimalarial drugs, chloroquine (CQ) and other 4-aminoquinolines have shown high potency and good bioavailability. Yet complications associated with drug resistance necessitate the discovery of effective new antimalarial agents. ADMET prediction studies were employed to evaluate a library of new molecules based on the 4-aminoquinolone-related structure of CQ. Extensive in vitro screening and in vivo pharmacokinetic studies in mice helped to identify two lead molecules, 18 and 4, with promising in vitro therapeutic efficacy, improved ADMET properties, low risk for drug-drug interactions, and desirable pharmacokinetic profiles. Both 18 and 4 are highly potent antimalarial compounds, with IC(50) values of 5.6 and 17.3 nM, respectively, against the W2 (CQ-resistant) strain of Plasmodium falciparum (for CQ, IC(50) = 382 nM). When tested in mice, these compounds were found to have biological half-lives and plasma exposure values similar to or higher than those of CQ; they are therefore desirable candidates to pursue in future clinical trials.

Effect of resveratrol on 17beta-estradiol sulfation by human hepatic and jejunal S9 and recombinant sulfotransferase 1E1.

The purpose of this study was to investigate the sulfation of resveratrol (3,5,4'-trihydroxystilbene) and its potential to exhibit drug-drug interactions via sulfation. The possible interaction of resveratrol with 17beta-estradiol (E2), a major estrogen hormone and prototypic substrate for sulfate conjugation, was studied. Resveratrol and E2 are both known to undergo sulfate conjugation catalyzed by human sulfotransferases (SULTs). Resveratrol is a phytoestrogen with mixed estrogen agonist/antagonist properties that is being developed as a chemopreventive agent. The sulfate conjugation of E2 and resveratrol were studied individually using S9 fractions from human liver and jejunum as well as recombinant human SULT isoforms. The sulfation of E2 (3-20 nM) was then investigated in the presence of various concentrations (0, 0.5, 1, and 2 microM) of resveratrol using the two S9 preparations as well as recombinant SULT1E1, the major isoform responsible for E2 sulfation. Resveratrol inhibited E2 sulfation with estimated K(i) values of 1.1 microM (liver), 0.6 microM (jejunum), and 2.3 microM (SULT1E1), concentrations that could be pharmacologically relevant. The results suggest that these phytoestrogens can potentially alter the homeostasis of estrogen levels. These findings also imply that resveratrol may inhibit the metabolism of other estrogen analogs or therapeutic agents such as ethinylestradiol or dietary components that are also substrates for SULT1E1.

Vesiculated alpha-tocopheryl succinate enhances the anti-tumor effect of dendritic cell vaccines.

Alpha tocopheryl succinate (alpha-TOS) is a non-toxic vitamin E analog under study for its anti-cancer properties. In an earlier study, we showed that alpha-TOS, when used in combination with non-matured dendritic cells (nmDC) to treat pre-established tumors, acts as an effective adjuvant. In this study, we have used vesiculated alpha-TOS (Valpha-TOS), a more soluble form of alpha-TOS that is relevant for clinical use, in combination with dendritic cells to treat pre-established murine tumors. We demonstrate that Valpha-TOS kills tumor cells in vitro and inhibits the growth of pre-established murine lung carcinoma (3LLD122) as effectively as alpha-TOS. The combination of Valpha-TOS plus non-matured or TNF-alpha-matured DC is more effective at inhibiting the growth of established tumors than Valpha-TOS alone. We also observed that Valpha-TOS induces expression of heat shock proteins in tumor cells and that co-incubation of non-matured DC with lysate derived from Valpha-TOS-treated tumor cells leads to DC maturation evidenced by up-regulation of co-stimulatory molecules and secretion of IL-12p70. This study therefore demonstrates the immunomodulatory properties of Valpha-TOS that may account for its adjuvant effect when combined with DC vaccines to treat established tumors.

alpha-Tocopheryl succinate sensitizes established tumors to vaccination with nonmatured dendritic cells.

PURPOSE: Dendritic cells (DCs) are considered potential candidates for cancer immunotherapy due to their ability to process and present antigens to T cells and stimulate immune responses. However, DC-based vaccines have exhibited minimal effectiveness against established tumors in mice and human cancer patients. The use of appropriate adjuvants can enhance the efficacy of DC-based cancer vaccines in treating established tumors. METHODS: In this study we have employed alpha-tocopheryl succinate (alpha-TOS), a nontoxic esterified analogue of vitamin E, as an adjuvant to enhance the effectiveness of DC vaccines in treating established murine Lewis lung (3LL) carcinomas. RESULTS: We demonstrate that locally or systemically administered alpha-TOS in combination with nonmatured DCs injected intratumorally (i.t.) or subcutaneously (s.c.) significantly inhibits the growth of preestablished 10-day tumors (mean tumor volume of 77.5 +/- 17.8 mm3 on day 30 post-tumor injection) as compared to alpha-TOS alone (mean tumor volume of 471 +/- 68 mm3 on day 30 post-tumor injection). Additionally, the adjuvant effect of alpha-TOS was superior to that of cyclophosphamide (CTX). The mean tumor volume on day 28 post-tumor injection in mice treated with CTX+DCs was 611 +/- 94 mm3 as compared to 105 +/- 36 mm3 in mice treated with alpha-TOS+DCs. Analysis of purified T lymphocytes from mice treated with alpha-TOS+DC revealed significantly increased secretion of IFN-gamma as compared to T cells from the various control groups. CONCLUSION: This study demonstrates the potential usefulness of alpha-tocopheryl succinate, an agent nontoxic to normal cell types, as an adjuvant to augment the effectiveness of DC-based vaccines in treating established tumors.

Subchronic oral toxicity of turmeric and ethanolic turmeric extract in female mice and rats.

Subchronic oral toxicity of turmeric and ethanolic turmeric extract was studied in female Swiss mice and Wistar rats fed turmeric (0, 1 and 5%) and ethanolic turmeric extract (0, 0.05 and 0.25%) through diet for 14 and/or 90 days. The administration of a high dose of turmeric (5%) for longer duration (90 days) showed a significant reduction in body weight gain, alterations in absolute and/or relative liver weights, and hepatotoxicity i.e. focal necrosis or focal necrosis with regeneration both in mice and rats. In mice lower doses of turmeric i.e 0.2 or 1% for 14 days also showed hepatotoxicity and they were found to be more vulnerable to turmeric-induced hepatotoxicity than rats.

Carcinogenicity studies of tobacco extract in vitamin A-deficient Sprague-Dawley rats.

Long-term carcinogenicity studies were carried out in male Sprague-Dawley rats maintained on vitamin A-sufficient (SLO+) and vitamin A-deficient (SLO-) diets and treated with tobacco extract (TE). Three-week-old rats received by gavage a total dose of 860 mg of TE at a daily dose of 3 mg/rat over a period of 21 months. Besides tumorigenicity, drug-metabolizing phase I and phase II enzymes in lung and liver as well as vitamin A and C levels in plasma and liver were measured at 12 and 21 months of age. The cumulative tumor incidence in TE-treated SLO- rats was significantly higher (77-100%) than that observed in TE-treated SLO+ rats (20-22%). Furthermore, SLO+ rats treated with TE showed lung and forestomach tumors, whereas TE-treated SLO- rats showed a preponderance of pituitary adenomas (87%). It was observed that TE treatment increased the activity of the hepatic and pulmonary phase I enzymes and decreased the glutathione/glutathione S-transferase detoxification system at both time points in SLO- rats. On TE treatment the vitamin A levels in the liver and plasma were significantly decreased with a concurrent increase in vitamin C levels. The data show that a vitamin A-deficient diet renders male Sprague-Dawley rats more susceptible to TE treatment than the vitamin A-sufficient diet, an effect which was associated with the augmented induction of P-450 content and activities and depletion of the glutathione/glutathione S-transferase pathway by TE.

Carcinogenicity studies of masheri: pyrolysed tobacco product, in vitamin-A-deficient Sprague Dawley rats.

The carcinogenicity of long-term feeding of masheri extract to animals in a vitamin-A-sufficient (SLO+) and deficient (SLO-) state was studied in Sprague Dawley rats by feeding daily dose of 3 mg extract over a period of 21 months. The phase I activating enzymes, the glutathione (GSH)/glutathione S-transferase (GST) detoxification system, and the hepatic and circulating levels of vitamins A and C were also monitored at 12 and 21 months. It was observed that the phase I enzyme activities were significantly higher in SLO+ than in SLO- rats at both 12 months and 21 months. Moreover, the SLO- masheri-treated animals also showed a decreased in the GSH/GST detoxification system while the reverse was observed in SLO+ group. Masheri extract treatment significantly lowered the hepatic and circulating levels of vitamin A while a concurrent increase was observed in the vitamin C level. The extract was found to be tumorigenic in both the SLO+ and SLO- groups. Benign tumours were observed in the SLO+ group while a high incidence of malignant tumours of the lung were observed in the SLO- group upon treatment with masheri extract.

Anticarcinogenic effect of betel leaf extract against tobacco carcinogens.

Epidemiological studies have implicated that betel quid offers some protection to tobacco induced carcinogenesis. Earlier studies in our laboratory have shown betel leaf extract (BLE) to be antimutagenic against standard mutagens and tobacco-specific N'-nitrosamines (TSNA), N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In the present study, we have tested the anticarcinogenic effect of BLE using Swiss male mice. Two protocols of study were used to test this effect. In the first protocol, the effect of BLE was tested against the standard carcinogen benzo[a]pyrene (BP) using Wattenberg's stomach tumor model, Cancer Res., 41 (1981) 2820-2823. In this protocol, BLE inhibited the tumorigenicity of BP to a significant extent. In the second protocol, the effect of BLE against the two tobacco-specific nitrosamines, NNN and NNK was studied using long-term studies on Swiss male mice. The nitrosamines were administered on the tongues of the mice, while the BLE was supplied in drinking water. Two doses of NNN (22 mg and 72 mg) and one dose of NNK (22 mg) were used. In this study, it was observed that the number of tumor bearing animals decreased, but the difference was significant only in the group treated with the low dose of NNN in combination with BLE. However, in all the BLE treated animals, irrespective of the dose of nitrosamine, the hepatic vitamin A and C levels were elevated significantly as compared to the corresponding nitrosamine-treated controls. These results indicate that BLE has a promising anticarcinogenic role to play in tobacco induced cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Weak carcinogenic effect of masheri, a pyrolysed tobacco product, in mouse skin tumor models.

Two commonly used varieties of masheri, pyrolysed tobacco products, were tested for their skin carcinogenicity in Swiss mice and the more sensitive strain of Swiss bare mice. In Swiss mice, painting of brown and black varieties of masheri extract did not show any tumorgenic effect; however, a marginal synergistic effect of 7,12-dimethylbenz[a]anthracene (DMBA) and black masheri extract was observed when DMBA was used as an initiator. In Swiss bare mice, black masheri extract induced tumors in 20%-35% animals at both the doses tested. In an initiation/promotion protocol with DMBA as an initiator, induction of tumors in 50%-52% Swiss bare mice and a slight synergistic effect of black masheri extract were observed with a low dose of DMBA, suggesting a synergistic effect.

Carcinogenicity studies of masheri, a pyrolysed product of tobacco.

The carcinogenicity of two commonly used brown and black varieties of masheri, a pyrolysed tobacco product, was studied by feeding the masheri through the diet at a 10% level to three different animal species of both sexes. In Sprague-Dawley rats, only brown masheri was used, while in Swiss mice and Syrian golden hamsters both varieties were used. In all the three species, forestomach papillomas were induced as a result of masheri treatment. In rats, 37% of animals showed forestomach papillomas while in mice and hamsters the incidence was 42-47% and 25-43%, respectively. No malignant changes were observed in any of the groups except 2/23 male hamsters showed forestomach carcinoma in the black masheri diet group.

Carcinogenicity of diethylnitrosamine in vitamin-A-deficient mice.

The effect of suboptimal levels of dietary vitamin A on diethylnitrosamine (DEN) carcinogenesis was studied in BALB/c and Swiss mice. Two different dietary regimens were employed to induce vitamin A deficiency and DEN was administered by gavage at 2 dose levels: 0.6 mg/kg as a single dose and 200 mg/kg in 4 divided doses. Shark liver oil (SLO) which was the main source of vitamin A in the standard diet, was deleted in one regimen and reduced to 25% in the other. The mice maintained on the former diet were given a high dose of DEN and those on the latter diet received a low dose. In both strains the deficient mice had a greater tumour incidence than those on standard diet with a marginal reduction in the latent period. At the low level of DEN there was shift in organotrophy, i.e. from liver in controls to lung in the vitamin-A-deficient mice of BALB/c strain. With the higher dose, lung adenomas predominated in deficient as well as control groups in both the strains. Forestomach carcinomas appeared in deficient mice and not in the controls.

Chilli extract treatment and induction of eye lesions in hamsters.

On chronic administration of 20 microliter of chilli extract to the cheek pouch of hamsters till death, 23% of the hamsters developed shrunken eye balls and closing of the eyelids. This effect was not observed in hamsters which received a single application of the potent carcinogen methylacetoxymethyl nitrosamine (DMN-OAC) (2 mg/kg body wt.) prior to repeated treatment with chilli extract. Vitamin A levels decreased significantly in the liver tissue of chilli-treated and carcinogen + chilli-treated groups compared to absolute alcohol-treated and untreated groups, while serum Vitamin A values decreased only in the DMN-OAC + chilli-treated group. However, Vitamin A levels do not seem to be linked causally with the effect on the eyes of chilli-treated hamsters, because these hamsters had circulating levels of Vitamin A comparable to those observed in untreated and alcohol-treated groups.