Pharmacokinetics of deguelin, a cancer chemopreventive agent in rats.

PURPOSE: To study the pharmacokinetics of deguelin, a naturally occurring potential cancer chemopreventive agent, in rats. METHODS: [3H]Deguelin was administered intravenously (i.v.) under anesthesia, and blood samples were collected over 24 h. [3H]Deguelin and metabolites were extracted from plasma with ethyl acetate, and quantified by HPLC. Data were analyzed with the WinNolin pharmacokinetic software package to determine pharmacokinetic parameters. A three-compartment first-order elimination model was used to fit the plasma concentration-time curve. In addition, deguelin concentrations in tissues after i.v. and intragastric (i.g.) administration were determined by HPLC, and excretion (feces and urine) was evaluated over a 5-day period after i.g. administration. RESULTS: Deguelin exhibited a mean residence time (MRT) of 6.98 h and terminal half-life (t1/2(gamma)) of 9.26 h. The area under the curve (AUC) and total clearance (Cl) were 57.3 ng.h/ml and 4.37 l/h per kg, respectively, with an apparent volume of distribution (V) and volume of distribution at steady-state (Vss) of 3.421 l/kg and 30.46 l/kg, respectively. Following i.v. administration, the relative levels of tissue distribution were as follows: heart > fat > mammary gland > colon > liver > kidney > brain > lung. Following i.g. administration, the relative levels of tissue distribution were as follows: perirenal fat > heart > mammary gland > colon > kidney > liver > lung > brain > skin. Within 5 days of i.g. administration, about 58.1% of the [3H]deguelin was eliminated via the feces and 14.4% via the urine. Approximately 1.7% of unchanged deguelin was found in the feces, and 0.4% in the urine. CONCLUSIONS: An initial pharmacokinetic investigation of deguelin showed that this rotenoid has a relatively long MRT and half-life in plasma in the rat. The compound distributed in the tissues and excreted as metabolites, mainly via the feces.

Cytotoxic sesquiterpenoids from Ratibida columnifera.

Bioassay-directed fractionation of the flowers and leaves of Ratibida columnifera using a hormone-dependent human prostate (LNCaP) cancer cell line led to the isolation of 10 cytotoxic substances, composed of five novel xanthanolide derivatives (2-4, 7, and 8), a novel nerolidol derivative (9), and three known sesquiterpene lactones, 9alpha-hydroxy-seco-ratiferolide-5alpha-O-angelate+ ++ (1), 9alpha-hydroxy-seco-ratiferolide-5alpha-O-(2-methylbut yrate) (5), 9-oxo-seco-ratiferolide-5alpha-O-(2-methylbutyrate) (6), as well as a known flavonoid, hispidulin (10). On the basis of its cytotoxicity profile, compound 5 was selected for further biological evaluation, and was found to induce G1 arrest and slow S traverse time in parental wild type p53 A2780S cells, but only G2/M arrest in p53 mutant A2780R cells, with strong apoptosis shown for both cell lines. The activity of 5 was not mediated by the multidrug resistance (MDR) pump, and it was not active against several anticancer molecular targets (i.e., tubulin polymerization/depolymerization, topoisomerases, and DNA intercalation). While these results indicate that compound 5 acts as a cytotoxic agent via a novel mechanism, this substance was inactive in in vivo evaluations using the murine lung carcinoma (M109) and human colon carcinoma (HCT116) models.

Pharmacokinetics and tissue distribution of betulinic acid in CD-1 mice.

Betulinic acid is a naturally occurring pentacyclic triterpenoid. Betulinic acid has recently been selected by the National Cancer Institute for addition into the RAID (Rapid Access to Intervention in Development) programme. The agent exhibits potential anti-tumour activity and functions in this regard via apoptosis. The objective of the present study was to determine the pharmacokinetics of betulinic acid in CD-1 mice. Serum samples were obtained at designed times after a single 250 or 500 mg/kg intraperitoneal (IP) dose of betulinic acid. Tissue samples (skin, heart, liver, spleen, kidney, lung, brain, colon, caecum, ovary, uterus, thymus, lymph node, bladder, perirenal fat, mammary gland and small intestine) were collected after betulinic acid administration (500 mg/kg). Betulinic acid was extracted with methylene chloride and quantitatively analysed by HPLC/MS. Oleanolic acid and madecassic acid were used as internal standards. Pharmacokinetic parameters were calculated using the WinNonlin pharmacokinetic software package. A two-compartment, first-order model was selected for pharmacokinetic modelling. The results showed that after IP 250 and 500 mg/kg betulinic acid, the serum concentrations reached peaks at 0.15 and 0.23 h, respectively. The 250 and 500 mg/kg above betulinic acid IP doses were found to have elimination half-lives of 11.5 and 11.8 h and total clearances of 13.6 and 13.5 L/kg/h, respectively. The pharmacokinetic parameters observed for IP betulinic acid 500 mg/kg in the skin of mice were as follows: k(a) (h(-1)) 0.257, k(10) (h(-1)) 0.234, t(1/2(alpha)) (h) 2.63, t(1/2(beta)) (h) 20.2, V (L/kg) 0.61, AUC (microg/h/mL) 3504, T(max) (h) 3.90 and C(max) (microg/mL) 300.9. The distribution of betulinic acid in tissues at 24 h post-IP administration in a descending order was as follows: perirenal fat, ovary, spleen, mammary gland, uterus, bladder, lymph node, liver, small intestine, caecum, lung, thymus, colon, kidney, skin, heart and brain.

Angoline and chelerythrine, benzophenanthridine alkaloids that do not inhibit protein kinase C.

Starting with an extract derived from the stem of Macleaya cordata (Papaveraceae) that was active in the process of inhibiting phorbol 12,13-dibutyrate binding to partially purified protein kinase C (PKC), the benzophenanthridine alkaloid angoline was isolated and identified. This discovery appeared in context, as a related benzophenanthridine alkaloid, chelerythrine, has been reported to mediate a variety of biological activities, including potent and selective inhibition of protein kinase C (PKC). However, in our studies, angoline was not observed to function as a potent inhibitor of PKC. Moreover, we were unable to confirm the reported inhibitory activity of chelerythrine. In a comprehensive series of studies performed with various PKC isozymes derived from a variety of mammalian species, neither chelerythrine nor angoline inhibited activity with high potency. To the contrary, chelerythrine stimulated PKC activity in the cytosolic fractions of rat and mouse brain in concentrations up to 100 microM. In addition, chelerythrine and angoline did not inhibit [3H]phorbol 12,13-dibutyrate binding to the regulatory domain of PKC at concentrations up to 40 microg/ml, and no significant alteration of PKC-alpha, -beta, or -gamma translocation was observed with human leukemia (HL-60) cells in culture. Further, chelerythrine did not inhibit 12-O-tetradecanoylphorbol 13-acetate-induced ornithine decarboxylase activity with cultured mouse 308 cells, but angoline was active in this capacity with an IC50 value of 1.0 microg/ml. A relatively large number of biological responses have been reported in studies conducted with chelerythrine, and alteration of PKC activity has been considered as a potential mechanism of action. In light of the current report, mechanisms independent of PKC inhibition should be considered as responsible for these effects.

Xanthones from the twigs of Mammea siamensis.

1,2-Dimethoxy-5-hydroxyxanthone, a new xanthone, was isolated from the twigs of Mammea siamensis, in addition to six known xanthones (5-hydroxy-1-methoxy-, 1,3-dimethoxy-5-hydroxy-, 2,5-dihydroxy-1-methoxy-, 1,7-dihydroxy-, 1,3,7-trihydroxy- and 3,5-dihydroxy-1-methoxyxanthone). Structures for these compounds were deduced from their spectral data.

Involvement of protein kinase and G proteins in the signal transduction of benzophenanthridine alkaloid biosynthesis.

We have previously reported that elicitor-induced benzophenanthridine alkaloid biosynthesis in suspension-cell cultures of Sanguinaria canadensis L. (SCP-GM) is mediated by a signal transduction system that involves calcium and possibly protein kinase(s). In this work, a number of exogenous agents were employed to further investigate the components of the signal transduction pathway involved in the induction of alkaloid biosynthesis by a fungal elicitor and abscisic acid (ABA). SCP-GM suspension-cells were treated with compounds that modify protein kinase activity, including phorbol esters, and 1-oleoyl-2-acetyl-rac-glycerol (OAG), a synthetic diacylglycerol analogue. Phorbol-12-myristate-13-acetate induced alkaloid accumulation by as much as 65-fold over control values, while the negative control, phorbol-13-monoacetate, had no effect. OAG also increased alkaloid production by approximately 25-fold as compared to controls. Likewise, pretreatment of the suspension-cell cultures with H-7 or staurosporine, significantly suppressed ABA- or fungal-induction of benzophenanthridine alkaloid biosynthesis. Modulators of GTP-binding protein activity were also active in this system. Treatment of the suspension-cells with cholera toxin (CHX) induced alkaloid accumulation by 25-fold, which increased to 34-fold when CHX was combined with a fungal elicitor derived from Penicillium expansum (PE), and 32-fold when CHX was combined with ABA. Treatment of SCP-GM cells with CHX also enhanced the activities of two N-methyltransferases in the benzophenanthridine biosynthetic pathway namely, tetrahydroberberine-N-methyltransferase and tetrahydrocoptisine-N-methyltransferase, by six and seven fold, respectively. Furthermore, benzophenanthridine alkaloid biosynthesis was induced by treating the suspension-cells with the G-protein activators, mastoparan, mas-7 or melittin, while the inactive homologue, mas-17, did not. Suppression of alkaloid accumulation occurred when the suspension-cells were treated with GDP beta S or pertussis toxin prior to treatment of the SCP-GM cells with either PE or ABA. The results support the hypothesis that one or more protein kinases, and putative G proteins are involved in the signal transduction pathway that mediates ABA and fungal-induced benzophenanthridine alkaloid biosynthesis.

Limonoids from Azadirachta excelsa.

Activity-directed fractionation of a stem extract of Azadirachta excelsa using KB (human oral epidermoid carcinoma) cells led to the isolation of four meliacin-type limonoids. Two of these constituents were novel, namely, 2,3-dihydronimbolide and 3-deoxymethylnimbidate, and these were purified along with the known compounds, nimbolide and 28-deoxonimbolide. The structures of the new compounds were determined by spectroscopic methods. Nimbolide and 28-deoxonimbolide were broadly cytotoxic when evaluated against a panel of human cancer cell lines, while the two novel compounds were inactive in this regard. The defection of nimbolide and 28-deoxonimbolide as cytotoxic constituents was facilitated by an electrospray LC/MS dereplication procedure.

Cancer chemopreventive activity of resveratrol, a natural product derived from grapes.

Resveratrol, a phytoalexin found in grapes and other food products, was purified and shown to have cancer chemopreventive activity in assays representing three major stages of carcinogenesis. Resveratrol was found to act as an antioxidant and antimutagen and to induce phase II drug-metabolizing enzymes (anti-initiation activity); it mediated anti-inflammatory effects and inhibited cyclooxygenase and hydroperoxidase functions (antipromotion activity); and it induced human promyelocytic leukemia cell differentiation (antiprogression activity). In addition, it inhibited the development of preneoplastic lesions in carcinogen-treated mouse mammary glands in culture and inhibited tumorigenesis in a mouse skin cancer model. These data suggest that resveratrol, a common constituent of the human diet, merits investigation as a potential cancer chemopreventive agent in humans.

Triterpenes from the combined leaf and stem of Lithospermum caroliniense.

An investigation of the combined leaf and stem of Lithospermum caroliniense afforded two new pentacyclic triterpenoids based on the olean-12-ene and taraxast-12-ene skeletal types. The structures of these compounds were elucidated on the basis of spectral analysis as 1 alpha,3 beta,23-trihydroxyolean-12-ene-28-oic acid and 3 alpha,19 beta,21 alpha,23-tetrahydroxytaraxast-12-ene-28-oic acid.

Two isoflavones from the bark of Petalostemon purpureus.

Two new isoflavones, 6,7,8,3',4',5'-hexamethoxyisoflavone and 7,8,3',4',5'-pentamethoxyisoflavone, have been isolated and characterized from the combined root bark and stem bark of Petalostemon purpureus.

A new prenylated flavonol from the root of Petalostemon purpureus.

A new prenylated flavonol, petalopurpurenol (1), and a known dihydroflavonol, petalostemumol (2), have been isolated by DNA scission-guided fractionation of the organic portion of a 20% MeOH/CHCl3/H2O partition of a 50% MeOH/CHCl3 extract of the roots of Petalostemon purpureus. Compound 2 displayed moderate activity in DNA-scission assay. Both compounds 1 and 2 were evaluated for cytotoxicity in a panel of human cancer cell lines. The structures of petalopurpurenol (1) and petalostemumol (2) were determined by spectroscopic analysis.

Two isoflavones from the bark of Petalostemon purpureus.

Two new isoflavones, 6,7,8,3',4',5'-hexamethoxyisoflavone and 7,8,3',4',5'-pentamethoxyisoflavone, have been isolated and characterized from the combined root bark and stem bark of Petalostemon purpureus.

Effect of tannins on screening of plant extracts for enzyme inhibitory activity and techniques for their removal.

Ethyl acetate and aqueous extracts of tannin-containing topoisomerase inhibitory plant samples were subjected to one or more of seven tannin removal procedures, and the resulting products were subsequently evaluated for topoisomerase inhibitory activity. In most of the samples investigated, the initial activity was lost after tannin removal. It was concluded that the activity initially observed was primarily due to tannins. Procedures are presented for routinely obtaining tannin-free organic and aqueous fractions.

Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis.

As a result of bioassay-guided fractionation, betulinic acid, a pentacyclic triterpene, was identified as a melanoma-specific cytotoxic agent. In follow-up studies conducted with athymic mice carrying human melanomas, tumour growth was completely inhibited without toxicity. As judged by a variety of cellular responses, antitumour activity was mediated by the induction of apoptosis. Betulinic acid is inexpensive and available in abundant supply from common natural sources, notably the bark of white birch trees. The compound is currently undergoing preclinical development for the treatment or prevention of malignant melanoma.

Cytotoxic biflavonoids from Selaginella willdenowii.

Bioactivity-guided fractionation of the leaves of Selaginella willdenowii afforded three known biflavones, 4',7"-di-O-methylamentoflavone, isocryptomerin and 7"-O-methylrobustaflavone, that were significantly cytotoxic against a panel of human cancer cell lines. Non-cytotoxic isolates were also obtained, namely, amentoflavone, bilobetin, robustaflavone and 2",3"-dihydroisocryptomerin, a new dihydrobiflavone. The structure for the new biflavonoid was unambiguously assigned by a combination of spectroscopic methods.

Quercetin-induced benzophenanthridine alkaloid production in suspension cell cultures of Sanguinaria canadensis.

Addition of micromolar concentrations of quercetin or rutin to suspension cell cultures of Sanguinaria canadensis L. (bloodroot) induced the biosynthesis of sanguinarine and chelerythrine in a dose-dependent manner. In contrast, related compounds: baicalein, naringin, naringenin, catechin, caffeic acid and benzoic acid displayed very weak inductive activity. Of the two active flavonoids, quercetin was the most effective for inducing benzophenanthridine alkaloid biosynthesis, with doses of 100 microM increasing alkaloid production over 375% as compared to negative controls. Quercetin's inductive effects were similar to that of an elicitor derived from fungus Penicillium expansum (PE-elicitor). Suppression of quercetin and PE-induced alkaloid biosynthesis by low doses of actinomycin D (5 micrograms/ml, alpha-amanitin (20 micrograms/ml), or cycloheximide (1 microgram/ml) demonstrate a requirement for both RNA and de novo cytoplasmic protein synthesis and suggest that alterations in gene expression are involved in the inductive mechanism. Furthermore, quercetin-induced alkaloid biosynthesis was significantly reduced by pretreatment of the cells with the calcium chelator, EGTA (3 mM), or the calcium channel inhibitor, verapamil (100 microM), suggesting that this process was calcium dependent.

Cancer preventive properties of varieties of Brassica oleracea: a review.

Cabbage, broccoli, Brussels sprouts, and other members of the genus Brassica have been widely regarded as potentially cancer preventative. This view is often based on both experimental testing of crude extracts and epidemiological data. The experimental evidence that provides support for this possibility is reviewed for the commonly consumed varieties of Brassica oleracea. In a majority of cases the biological activities seen in testing crude extracts may be directly related to specific chemicals that have been reported to be isolated from one of these closely related species, thus the chemical evidence further supports the data from testing extracts and epidemiology.

Novel antimitotic dibenzocyclo-octadiene lignan constituents of the stem bark of Steganotaenia araliacea.

By means of activity-directed chromatographic fractionation using cultured astrocytoma (ASK) cells, six dibenzocyclo-octadiene lignans were isolated from Steganotaenia araliacea stem bark. In addition to the most abundant analogue, steganangin [1], two other known compounds, steganacin [3] and steganolide A [6], and three new compounds, episteganangin [2], steganoate A [4], and steganoate B [5], were obtained. Episteganangin [2] was chemically correlated with the known ketone steganone [7]. All of these compounds demonstrated cytotoxic activity when tested against a panel of eleven human tumor cell lines, with the exception of steganoate A [4]. The magnitude of this activity tended to correlate with antimitotic activity observed with the ASK assay and in vitro inhibition of microtubule assembly. Steganacin [3] was less cytotoxic than colchicine, but more active in these latter two assay systems.

Isolation of echinocystic acid-3-O-sulfate, a new triterpene, from Tetrapleura tetraptera, and evaluation of the mutagenic potential of molluscicidal extracts and isolates.

A known triterpene glycoside, 3-O-[beta-D-glucopyranosyl-(1"-6')-2'-acetamido-2'-deoxy-beta-D-gluco pyranosyl]olean-12-en-28-oic acid [3], and new sulfated triterpene, echinocystic acid-3-O-sodium sulfate [4], have been isolated from the stem bark of Tetrapleura tetraptera. Compound 3 was 100% lethal to Biomphalaria glabrata at 20 ppm, while 4 was not molluscicidal at the same concentration. In a forward mutation assay utilizing Salmonella typhimurium strain TM677, T. tetraptera stem bark extracts were found to be mutagenic in the absence of a metabolic activating system (S-9). An MeOH extract of the fruit exhibited weak mutagenic activity only in the presence of S-9. The stem bark isolates, which included aridanin [1], 3-O-(2'-acetamido-2'-deoxy-beta-D-glucopyranosyl)echinocystic acid [2], and compounds 3 and 4, were not mutagenic either with or without metabolic activation.