Oral toxicity and pharmacokinetic studies of SHetA2, a new chemopreventive agent, in rats and dogs.

SHetA2 is a heteroarotinoid that has shown selective inhibition of cancer cell growth and an induction of apoptosis without activation of nuclear retinoic acid receptors. In the rat study, SHetA2 was administered in 1% aqueous methylcellulose/0.2% Tween 80 by oral gavage at 0, 100, 500, and 2,000 mg/kg/day for 28 days. The high-dose administration induced decreased activity in male rats, decreased body-weight gains and food consumption, and changes in organ weights. The major metabolite of SHetA2 in rat plasma was monohydroxy SHetA2, which was considerably higher than the parent compound after oral and intravenous administration. Pharmacokinetic analysis showed extremely low (<1%) systemic bioavailability of SHetA2 for all doses tested. The dose of 2,000 mg/kg/day was considered as the lowest observed adverse effect level. The no observed adverse effect level (NOAEL) was 500 mg/kg/day. In the dog study, no toxicity of SHetA2 in 30% aqueous Solutol(®) HS 15 was observed in any tested dose groups (0, 100, 400, and 1,500 mg/kg/day). The major metabolite of SHetA2 in dog plasma was also monohydroxy SHetA2, which was equal to or lower than the parent compound after oral administration. SHetA2 levels in dog plasma were notably higher, when compared to levels in rat plasma. However, exposure was not dose proportional, as exemplified by a lack of proportional increase in maximum concentration or area under the plasma concentration-time curve with increasing dose. The NOAEL was not established and was considered to be above 1,500 mg/kg/day.

Genotoxicity of the cancer chemopreventive drug candidates CP-31398, SHetA2, and phospho-ibuprofen.

The genotoxic activities of three cancer chemopreventive drug candidates, CP-31398 (a cell permeable styrylquinazoline p53 modulator), SHetA2 (a flexible heteroarotinoid), and phospho-ibuprofen (PI, a derivative of ibuprofen) were tested. None of the compounds were mutagenic in the Salmonella/Escherichia coli/microsome plate incorporation test. CP-31398 and SHetA2 did not induce chromosomal aberrations (CA) in Chinese hamster ovary (CHO) cells, either in the presence or absence of rat hepatic S9 (S9). PI induced CA in CHO cells, but only in the presence of S9. PI, its parent compound ibuprofen, and its moiety diethoxyphosphoryloxybutyl alcohol (DEPBA) were tested for CA and micronuclei (MN) in CHO cells in the presence of S9. PI induced CA as well as MN, both kinetochore-positive (Kin+) and -negative (Kin-), in the presence of S9 at ≤100µg/ml. Ibuprofen was negative for CA, positive for MN with Kin+ at 250µg/ml, and positive for MN with Kin- at 125 and 250µg/ml. DEPBA induced neither CA nor MN at ≤5000µg/ml. The induction of chromosomal damage in PI-treated CHO cells in the presence of S9 may be due to its metabolites. None of the compounds were genotoxic, in the presence or absence of S9, in the GADD45α-GFP Human GreenScreen assay and none induced MN in mouse bone marrow erythrocytes.

Assessment of oral toxicity and safety of 9-cis-UAB30, a potential chemopreventive agent, in rat and dog studies.

9-cis-UAB30 is a potential chemopreventative agent that has been shown to be effective on many different types of tumors. The safety and toxicity of 9-cis-UAB30 had not been previously established. These studies were conducted to evaluate the potential toxicity and pharmacokinetics in a rodent and a nonrodent species for the purpose of investigational new drug submission. Oral gavage administration of 9-cis-UAB30 at the doses 0, 3, 15, and 100 mg/kg/day to CD® rats for 28 days showed a dose-dependent (although not dose-proportional) increase in plasma drug levels in week 4. The liver was the target organ for toxicity of 9-cis-UAB30. Hepatomegaly along with increases in serum aspartate-aminotransferase and alkaline-phosphatase levels were seen in rats. Moderate hypoalbuminemia and hyperglobulinemia resulted in a decreased albumin/globulin ratio. Histopathology revealed hepatocellular change consistent with hepatic glycogen deposition. Toxicity studies in dogs did not show treatment-related toxicity at doses as high as 100 mg/kg/day (highest dose tested) administered by capsules for 28 days. No effects on the central nervous system (functional observational battery in rats) or cardiovascular function (safety pharmacology study in telemeterized dogs) were seen. The no observed adverse effect level (NOAEL) in the rat studies was 3 mg/kg/day; however, the adverse effects seen in rats receiving 15 mg/kg/day (the least observed adverse effect level) was a slight, but statistically significant, elevation in fibrinogen and decrease in prothrombin time, which may be a sign of some tendency for increased blood coagulation. The NOAEL in the dog study was at least 100 mg/kg/day.

Murine oncogenicity and pharmacokinetics studies of 9-cis-UAB30, an RXR agonist, for breast cancer chemoprevention.

The synthetic retinoic acid analog, 9-cis-UAB30 [(2E,4E,6Z,8E)-8-(3',4'-dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acid], is a specific ligand for the retinoid X receptor. Murine oncogenicity and pharmacokinetics studies were performed as part of the preclinical development of 9-cis-UAB30 for breast cancer chemoprevention. In the oncogenicity study, TSG-p53((+/-)) (p53 knockout) mice (25 per sex per group) received daily gavage exposure to 9-cis-UAB30 doses of 0 (control), 30, 100, or 300 mg/kg/d for 6 months. Positive controls received p-cresidine (400 mg/kg/d) for 6 months. 9-cis-UAB30 had no biologically significant effects on survival, body weight, body weight gain, clinical signs, hematology, or clinical chemistry but induced dose-related hepatomegaly in both sexes and decreased thymus weights in high-dose females. Gross and microscopic pathology provided no evidence of 9-cis-UAB30 toxicity or oncogenicity; by contrast, p-cresidine induced urinary bladder neoplasms in more than 60% of male and female mice. It was concluded that 9-cis-UAB30 is not oncogenic in p53((+/-)) mice. In the pharmacokinetics study, C57BL/6 mice received daily gavage exposure to 9-cis-UAB30 (100 or 300 mg/kg/d) for 1 or 7 days. Pharmacokinetic parameters were similar after 1 and 7 days of dosing. Dose-related peak plasma levels of 9-cis-UAB30 were seen between 0.25 and 3 hours; volume of distribution was comparable at both dose levels. Increases in area under the curve were less than proportional to dose and were associated with an increased rate of apparent clearance and decreased elimination half-life. These results suggest decreased absorption and/or possible induction of clearance mechanisms. Enzyme induction may underlie the hepatomegaly seen in mice treated with 9-cis-UAB30 for 6 months in the oncogenicity study.

Identification of [14C]fluasterone metabolites in urine and feces collected from dogs after subcutaneous and oral administration of [14C]fluasterone.

The objective of this research was the identification of the metabolic profile of fluasterone, a synthetic derivative of dehydroepiandrosterone, in dogs treated orally or subcutaneously with [4-(14)C]fluasterone. Separation and characterization techniques used to identify the principal metabolites of fluasterone in urine and feces included high-performance liquid chromatography (HPLC), liquid scintillation spectrometry, HPLC/tandem mass spectrometry, and NMR. In urine, the majority of the radioactivity was present as two components that had apparent molecular weights consistent with their tentative identification as monoglucuronide conjugates of 4alpha-hydroxy-16alpha-fluoro-5-androsten-17beta-ol and X(alpha or beta)-4alpha-dihydroxy-16alpha-fluoro-5-androsten-17beta-ol. The identification of the monoglucuronide conjugate of 4alpha-hydroxy-16alpha-fluoro-5-androsten-17beta-ol was also supported by NMR data. In support of this identification, these metabolites were cleaved with glucuronidase enzyme treatment, which gave rise to components with molecular weights again consistent with the aglycones of a monohydroxylated, 17-keto reduced (dihydroxy) fluasterone metabolite and a dihydroxylated, 17-keto reduced (trihydroxy) fluasterone metabolite. In feces, nonconjugated material predominated. The primary metabolites eliminated in feces were the two hydroxy fluasterone metabolites arising from 17-reduction (16alpha-fluoro-5-androsten-17beta-ol and 16alpha-fluoro-5-androsten-17alpha-ol) and 4alpha-hydroxy-16alpha-fluoro-5-androsten-17beta-ol that was present in urine in glucuronide form.

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.

Preventive effects of polyphenon E on urinary bladder and mammary cancers in rats and correlations with serum and urine levels of tea polyphenols.

Polyphenon E, a standardized mixture of green tea polyphenols, was examined for its chemopreventive efficacy against chemically induced urinary bladder and mammary cancers. In the present study, Polyphenon E was administered after the last dose of 4-hydroxybutyl(butyl)nitrosamine, or roughly 30% of the way into the experiment. Polyphenon E (100 or 250 mg/kg body weight/d) caused a dose-dependent decrease in palpable urinary bladder tumors [low dose, 14 of 34; high dose, 6 of 35; controls, 20 of 34 (P < 0.01)]. In the mammary cancer model, Polyphenon E [333 or 1,000 mg/kg body weight (BW)/d] was administered beginning 5 days after a single dose of methylnitrosourea. In contrast to its significant efficacy in bladder tumor prevention, Polyphenon E had a minimal effect in the prevention of mammary cancers. Levels of polyphenols were determined in the urine and serum of rats. Relatively high levels of various polyphenols (and metabolites) were found in the urine. However, virtually no epigallocatechin-3-gallate was observed in the urine because of low systemic bioavailability; although it represents almost 65% of the polyphenols in Polyphenon E. Levels of polyphenols in serum were 50 x to 1,000 x less than were observed in urine. The bioavailability of these tea polyphenols to different organ sites may contribute to the differing preventive efficacy of Polyphenon E against urinary bladder and mammary cancers.

Subchronic toxicity and toxicogenomic evaluation of tamoxifen citrate + bexarotene in female rats.

Tamoxifen (TAM) is a nonsteroidal antiestrogen that prevents estrogen receptor-positive breast cancer in rodents and humans. Bexarotene (BEX), a selective agonist for retinoid X receptors, inhibits mammary carcinogenesis in rodents. The present study was conducted to support the preclinical development of TAM (tamoxifen citrate) + BEX for use in breast cancer chemoprevention, and to investigate the influence of these agents on hepatic gene expression. Female CD rats (20 per group) received daily oral (gavage) exposure to TAM (0 or 60 microg/kg/day) and/or BEX (0, 5, 15, or 45 mg/kg/day) for a minimum of 90 days. BEX induced mild, dose-related anemia and dose-related increases in serum alkaline phosphatase, cholesterol, triglycerides, and calcium levels, and increased platelet counts. TAM had no biologically significant effect on any clinical pathology parameter and did not alter the effects of BEX on these endpoints. Microscopic alterations induced by BEX included epidermal hyperplasia, hyperkeratosis (stomach), and cytoplasmic clearing (liver). Microscopic changes in TAM-treated rats were limited to mucous cell hypertrophy in the cervix and vagina. The toxicity of administration of the combination of TAM + BEX can generally be predicted on the basis of the toxicity of each drug as a single agent. BEX induced dose-related alterations in the expression of several genes involved in steroid, drug, and/or fatty acid metabolism; TAM did not alter these effects of BEX. Differential expression of genes involved in drug and lipid metabolism may underlie the observed effects of BEX on cholesterol and triglyceride levels and its effects on liver histology.

Evaluation of chemopreventive agents for genotoxic activity.

We conducted genetic toxicity evaluations of 11 candidate chemopreventive agents with the potential for inhibiting carcinogenesis in humans at increased risk of cancer. The compounds were evaluated for bacterial mutagenesis in the Salmonella-E. coli assay, for mammalian mutagenesis in mouse lymphoma cells, for chromosome aberrations in Chinese Hamster Ovary (CHO) cells, and for micronucleus induction in mouse bone marrow. Tested agents were indole 3-carbinol (I3C), bowman-birk inhibitor concentrate (BBIC), black tea polyphenols (BTP), farnesol, geraniol, l-Se-methylselenocysteine (SeMC), 5,6-dihydro-4H-cyclopenta[1,2]-dithiol-3-thione(DC-D3T), 4'-bromoflavone, 2,5,7,8-tetramethyl-(2R-[4R,8R,12-trimethyltridecyl] chroman-6-yloxy) acetic acid (alpha-TEA), SR13668 (2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo[2,3-b] carbazole and SR16157 (3-O-sulfamoyloxy-7alpha-methyl-21-(2-N,N-diethylaminoethoxy)-19-norpregna-1,3,5(10)-triene). All these agents, except I3C and BTP, were negative in the Salmonella-E. coli assay in the presence and absence of metabolic activation (S9). I3C and BTP induced a weak mutagenic response in the presence and absence of S9 with strains TA100 and TA98, respectively. Of the three compounds tested in the mouse lymphoma assay (I3C, BBIC, and BTP), only BTP was mutagenic in the presence of S9. In the chromosomal aberration assay, of the 8 compounds that were tested, 4'-bromoflavone elicited a positive response in the absence of S9 only, while SR16157 was positive in the presence of S9. The results with geraniol remain inconclusive. I3C, BBIC and BTP were not tested in the chromosomal aberration assay. None of the 11 agents induced micronuclei in mouse bone marrow erythrocytes.

Preliminary safety evaluation of the putative cancer chemopreventive agent tricin, a naturally occurring flavone.

PURPOSE: Naturally occurring flavonoids such as quercetin and genistein possess cancer chemopreventive properties in experimental models. However, adverse effects such as their mutagenicity confound their potential clinical usefulness. Furthermore in leukaemia cells some flavonoids cleave the breakpoint cluster region of the mixed lineage leukaemia (MLL) gene as a consequence of inhibition of topoisomerase II. The choice of flavonoids to be developed as cancer chemopreventive agents depends crucially on their safety. Here, we explored safety aspects of the novel flavone tricin, a constituent of rice bran and other grass species, which has recently been found to interfere with murine gastrointestinal carcinogenesis. METHODS: Evidence of pathological or morphological changes in liver, lung, heart, spleen, kidney, adrenal gland, pancreas or thymus tissues was studied in mice which received tricin, genistein or quercetin 1,000 mg/kg daily by the oral route on five consecutive days. The ability of tricin (50 microM) to cleave the MLL gene was studied in human leukaemia cells by Southern blotting, and its effect on human topoisomerase II activity was investigated in incubations with supercoiled DNA. The mutagenicity of tricin was assessed in the Salmonella/Escherichia coli assay, and its clastogenicity was adjudged by chromosomal aberrations in Chinese hamster ovary cells and occurrence of micronuclei in bone marrow erythrocytes in Swiss-Webster mice. RESULTS: Neither tricin, quercetin, or genistein caused pathological or morphological changes in any of the murine tissues studied. Tricin (50 microM) failed to cause MLL gene breakage, and it inhibited topoisomerase II only at 500 microM, but not at 10, 50 or 100 microM. Tricin lacked genotoxic properties in the systems studied here. CONCLUSION: The results tentatively suggest that tricin may be considered safe enough for clinical development as a cancer chemopreventive agent.

Glucuronidation of trans-resveratrol by human liver and intestinal microsomes and UGT isoforms.

Resveratrol (trans-resveratrol, trans-3,5,4'-trihydroxystilbene) is a naturally occurring stilbene analogue found in high concentrations in red wine. There is considerable research interest to determine the therapeutic potential of resveratrol, as it has been shown to have tumour inhibitory and antioxidant properties. This study was performed to investigate the glucuronidation of resveratrol and possible drug interactions via glucuronidation. Two glucuronide conjugates, resveratrol 3-O-glucuronide and resveratrol 4'-O-glucuronide, were formed by human liver and intestinal microsomes. UGT1A1 and UGT1A9 were predominantly responsible for the formation of the 3-O-glucuronide (Km = 149 microM) and 4'-O-glucuronide (Km = 365 microM), respectively. The glucuronide conjugates were formed at higher levels (up to 10-fold) by intestinal rather than liver microsomes. Resveratrol was co-incubated with substrates of UGT1A1 (bilirubin and 7-ethyl-10-hydroxycamptothecin (SN-38)) and UGT1A9 (7-hydroxytrifluoromethyl coumarin (7-HFC)). No major changes were noted in bilirubin glucuronidation in the presence of resveratrol. Resveratrol significantly inhibited the glucuronidation of SN-38 (Ki = 6.2 +/- 2.1 microM) and 7-HFC (Ki = 0.6 +/- 0.2 microM). Hence, resveratrol has the potential to inhibit the glucuronidation of concomitantly administered therapeutic drugs or dietary components that are substrates of UGT1A1 and UGT1A9.

Simultaneous determination of myristyl nicotinate, nicotinic acid, and nicotinamide in rabbit plasma by liquid chromatography-tandem mass spectrometry using methyl ethyl ketone as a deproteinization solvent.

Myristyl nicotinate (Nia-114) is an ester prodrug being developed for delivery of nicotinic acid (NIC) into the skin for prevention of actinic keratosis and its progression to skin cancer. To facilitate dermal studies of Nia-114, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using methyl ethyl ketone (MEK) as a deproteinization solvent was developed and validated for the simultaneous determination of Nia-114, NIC, and nicotinamide (NAM) in rabbit plasma. NAM is the principal metabolite of NIC, which is also expected to have chemopreventive properties. The analytes were chromatographically separated using a Spherisorb Cyano column under isocratic conditions, and detected by multiple reaction monitoring (MRM) in positive-ion electrospray ionization mode with a run time of 9 min. The method utilized a plasma sample volume of 0.2 ml and isotope-labeled D4 forms of each analyte as internal standards. The method was linear over the concentration range of 2-1000, 8-1000, and 75-1000 ng/ml, for Nia-114, NIC, and NAM, respectively. The intra- and inter-day assay accuracy and precision were within +/-15% for all analytes at low, medium, and high quality control standard levels. The relatively high value for the lower limit of quantitation (LLOQ) of NAM was demonstrated to be due to the high level of endogenous NAM in the rabbit plasma (about 350 ng/ml). Endogenous levels of NIC and NAM in human, dog, rat, and mouse plasma were also determined, and mean values ranged from <2 ng/ml NIC and 38.3 ng/ml NAM in human, to 233 ng/ml NIC and 622 ng/ml NAM in mouse. Nia-114 was generally unstable in rabbit plasma, as evidenced by loss of 44-50% at room temperature by 2 h, and loss of 64-70% upon storage at -20 degrees C for 1 week, whereas it was stable (<7% loss) upon storage at -80 degrees C for 1 month.

Modulation of hepatic and renal drug metabolizing enzyme activities in rats by subchronic administration of farnesol.

Farnesol demonstrates antitumor activity in several animal models for human cancer and was being considered for development as a cancer chemopreventive agent. This study was performed to characterize the effects of minimally toxic doses of farnesol on the activity of phase I and II drug metabolizing enzymes. CD((R)) rats (20/sex/group) received daily gavage exposure to farnesol doses of 0, 500, or 1000 mg/kg/day for 28 days; 10 rats/sex/group were necropsied at the termination of farnesol exposure; remaining animals were necropsied after a 28-day recovery period. No deaths occurred during the study, and farnesol had no significant effects on body weight, food consumption, clinical signs, or hematology/coagulation parameters. Modest but statistically significant alterations in several clinical chemistry parameters were observed at the termination of farnesol exposure; all clinical pathology effects were reversed during the recovery period. At the termination of dosing, the activities of CYP1A, CYP2A1-3, CYP2B1/2, CYP2C11/12, CYP2E1, CYP3A1/2, CYP4A1-3, CYP19, glutathione reductase, NADPH/quinone oxidoreductase and UDP-glucuronosyltransferase were significantly increased in the livers of farnesol-treated rats; farnesol also increased the activity of glutathione S-transferase in the kidney. The effects of farnesol on hepatic and renal enzymes were reversed during the recovery period. At the end of the dosing period, increases in absolute and relative liver and kidney weights were seen in farnesol-treated rats. These increases may be secondary to induction of drug metabolizing enzymes, since organ weight increases were not associated with histopathologic alterations and were reversed upon discontinuation of farnesol exposure. Administration of farnesol at doses of up to 1000 mg/kg/day induced reversible increases in the activities of several hepatic and renal drug metabolizing enzymes in rats, while inducing only minimal toxicity. It is concluded that non-toxic or minimally toxic doses of farnesol could alter the metabolism, efficacy, and/or toxicity of drugs with which it is co-administered.

Proceedings: the Applications of Bioinformatics in Cancer Detection Workshop.

The Division of Cancer Prevention of the National Cancer Institute sponsored and organized the Applications of Bioinformatics in Cancer Detection Workshop on August 6-7, 2002. The goal of the workshop was to evaluate the state of the science of bioinformatics and determine how it may be used to assist early cancer detection, risk identification, risk assessment, and risk reduction. This paper summarizes the proceedings of this conference and points out future directions for research.

Overview of commonly used bioinformatics methods and their applications.

Bioinformatics, in its broad sense, involves application of computer processes to solve biological problems. A wide range of computational tools are needed to effectively and efficiently process large amounts of data being generated as a result of recent technological innovations in biology and medicine. A number of computational tools have been developed or adapted to deal with the experimental riches of complex and multivariate data and transition from data collection to information or knowledge. These include a wide variety of clustering and classification algorithms, including self-organized maps (SOM), artificial neural networks (ANN), support vector machines (SVM), fuzzy logic, and even hyphenated techniques as neuro-fuzzy networks. These bioinformatics tools are being evaluated and applied in various medical areas including early detection, risk assessment, classification, and prognosis of cancer. The goal of these efforts is to develop and identify bioinformatics methods with optimal sensitivity, specificity, and predictive capabilities.

Low-calcemic, efficacious, 1alpha,25-dihydroxyvitamin D3 analog QW-1624F2-2: calcemic dose-response determination, preclinical genotoxicity testing, and revision of A-ring stereochemistry.

Based on an X-ray crystal structure determination, the A-ring stereochemistry of hybrid analog QW-1624F2-2 (1alpha-hydroxymethyl-16-ene-24,24-difluoro-25-hydroxy-26,27-bis-homovitamin D3) is revised to be 1alpha-CH2OH-3beta-OH. This analog is shown to be approximately 80-100 times less calciuric than the natural hormone 1alpha,25-dihydoxyvitamin D3. This analog is shown also to be non-genotoxic in three different standard assays.

Reactivity of atropaldehyde, a felbamate metabolite in human liver tissue in vitro.

Antiepileptic therapy with a broad spectrum drug felbamate (FBM) has been limited due to reports of hepatotoxicity and aplastic anemia associated with its use. It was proposed that a bioactivation of FBM leading to formation of alpha,beta-unsaturated aldehyde, atropaldehyde (ATPAL) could be responsible for toxicities associated with the parent drug. Other members of this class of compounds, acrolein and 4-hydroxynonenal (HNE), are known for their reactivity and toxicity. It has been proposed that the bioactivation of FBM to ATPAL proceeds though a more stable cyclized product, 4-hydroxy-5-phenyltetrahydro-1,3-oxazin-2-one (CCMF) whose formation has been shown recently. Aldehyde dehydrogenase (ALDH) and glutathione transferase (GST) are detoxifying enzymes and targets for reactive aldehydes. This study examined effects of ATPAL and its precursor, CCMF on ALDH, GST and cell viability in liver, the target tissue for its metabolism and toxicity. A known toxin, HNE, which is also a substrate for ALDH and GST, was used for comparison. Interspecies difference in metabolism of FBM is well documented, therefore, human tissue was deemed most relevant and used for these studies. ATPAL inhibited ALDH and GST activities and led to a loss of hepatocyte viability. Several fold greater concentrations of CCMF were necessary to demonstrate a similar degree of ALDH inhibition or cytotoxicity as observed with ATPAL. This is consistent with CCMF requiring prior conversion to the more proximate toxin, ATPAL. GSH was shown to protect against ALDH inhibition by ATPAL. In this context, ALDH and GST are detoxifying pathways and their inhibition would lead to an accumulation of reactive species from FBM metabolism and/or metabolism of other endogenous or exogenous compounds and predisposing to or causing toxicity. Therefore, mechanisms of reactive aldehydes toxicity could include direct interaction with critical cellular macromolecules or indirect interference with cellular detoxification mechanisms.

Enhanced oral bioavailability of the hydrophobic chemopreventive agent (SR13668) in beagle dogs.

Potency and activity of SR13668 in cancer prevention have been proven in several in vitro and in vivo cancer models. However, the compound is highly hydrophobic and its limited oral bioavailability has hindered its clinical translation. In this study, we encapsulated SR13668 into polymeric nanoparticles to increase compound aqueous solubility and therefore bioavailability. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (100-200 nm) encapsulating SR13668 with narrow size distribution and high drug loading were generated by a continuous and scalable process of flash nanoprecipitation integrated with spray dry. A single gavage dose of SR13668-PLGA nanoparticles at 2.8 mg/kg was administered in eight beagle dogs. Drug levels in animal whole blood and plasma were measured over 24 hours. Enhanced bioavailability of SR13668 using nanoparticles compared with formulations of Labrasol® and neat drug in 0.5% methylcellulose is reported. This is the first attempt to study pharmacokinetics of SR13668 in large animals with orally administrated nanoparticle suspension.

Determination of resveratrol and its sulfate and glucuronide metabolites in plasma by LC-MS/MS and their pharmacokinetics in dogs.

An analytical approach for the determination of trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene) and its glucuronide and sulfate conjugates in dog plasma by LC-MS/MS (without enzymatic hydrolysis of the conjugates) was validated to support pre-clinical toxicological and pharmacological studies. The approach required two independent sample extractions and consequent instrument runs. Samples for resveratrol determination were prepared by protein precipitation with acetonitrile; acetonitrile-methanol was used instead for resveratrol metabolites. Chromatographic separation was performed using a C18 column (30 mm × 2.0 mm) at a flow rate of 0.25 mL/min. For resveratrol the mobile phase consisted of A: 5mM ammonium acetate in water-isopropanol (98:2, v/v) and B: methanol-isopropanol (98:2, v/v) and for metabolites the mobile phase was modified as follows: A: 0.1% (v/v) formic acid in water and B: 0.1% (v/v) formic acid in acetonitrile. Total run time was 12 min for each run with retention times of about 4-5 min for all analytes. A turbo ion spray source was used operating in negative mode for resveratrol and resveratrol sulfate and in positive mode for resveratrol glucuronide. Calibration curves were linear from 5 to 1000 ng/mL for resveratrol and its glucuronide, and 10-2000 ng/mL for resveratrol sulfate. Linearity was assessed using the internal standard method for resveratrol and the external standard method for the metabolites. Method accuracy was 90-112% of the true value for all analytes with precision of 9% RSD or less for all validation experiments. The validated method was applied to a preclinical toxicology study in dogs after oral administration (200-1200 mg/kg) of the agent. Peak plasma resveratrol concentration (C(max)) for most animals was observed within 1-5 h of dosing, with group mean values in the 1.7-9.9 µg/mL (7.5-43 µM) range. Area under the plasma concentration-time curve (AUC) mean values for resveratrol ranged from 3.6 to 44 h µg/mL for all study groups and were generally proportional to the dose, with no consistent statistically significant changes observed for gender or number of doses. Mean molecular-weight adjusted ratios of resveratrol metabolites to resveratrol for AUC ranged from 1 to 9 for resveratrol glucuronide and from 2 to 11 for resveratrol sulfate.