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.

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.

Enhanced oral bioavailability of a cancer preventive agent (SR13668) by employing polymeric nanoparticles with high drug loading.

SR13668 [2,10-Dicarbethoxy-6-methoxy-5,7-dihydro-indolo-(2,3-b)carbazole] has been proven effective in cancer prevention, but the limited bioavailability has hindered its clinical translation. In this study, we have developed a continuous, scalable process to form stable poly(lactic-co-glycolic acid) nanoparticles encapsulating SR13668, based on understanding of the competitive kinetics of nanoprecipitation and spray drying. The optimized formulation achieved high drug loading (33.3 wt %) and small particles (150 nm) with narrow size distribution. The prepared nanoparticle suspensions through flash nanoprecipitation were spray dried to achieve long-term stability and to conveniently adjust the nanoparticle concentration before use. In vitro release of SR13668 from the nanosuspensions was measured in a solution with separated organic and aqueous phases to overcome the limit of SR13668 low water solubility. Higher oral bioavailability of SR13668 by employing polymeric nanoparticles compared with the Labrasol® formulation was demonstrated in a mouse model.

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.

Effects of bacterial and presystemic nitroreductase metabolism of 2-chloro-5-nitro-N-phenylbenzamide on its mutagenicity and bioavailability.

2-Chloro-5-nitro-N-phenylbenzamide (GW9662), a potent irreversible PPAR-γ antagonist, has shown promise as a cancer chemopreventive agent and is undergoing preclinical evaluations. Studies were initiated to assess its bacterial mutagenicity and pharmacokinetic profile in two animal species prior to subchronic oral toxicity evaluations and the results are reported here. GW9662 was mutagenic in both TA98 and TA100 bacterial strains with and without metabolic activation but was negative in the nitroreductase-deficient strains (TA98NR and TA100NR) also with and without metabolic activation, indicating that GW9662 mutagenicity is dependent on nitroreduction. The mutagenic activity was predominantly via a base-substitution mechanism. Following oral dosing in rats and dogs, the parent compound, GW9662, was virtually absent from plasma samples, but there was chromatographic evidence for the presence of metabolites in the plasma as a result of oral dosing. Metabolite identification studies showed that an amine metabolite ACPB (5-amino-2-chloro-N-phenylbenzamide), a product of nitro reduction, was the predominant species exhibiting large and persistent plasma levels. Thus systemic circulation of GW9662 has been attained largely in the form of its reduced metabolite, probably a product of gut bacterial metabolism. GW9662 was detectable in plasma of rats and dogs after intravenous dose albeit at low concentrations. Pharmacokinetic analysis following intravenous dosing in rats showed a rapid clearance and an extensive tissue distribution which could have accounted for the very low plasma levels. Of note, the amine metabolite was absent following intravenous dosing in both rats and dogs, confirming it being a product of presystemic metabolism. The potential utility of GW9662 as a chemopreventive agent, especially as an Estrogen Receptor-α (ER-α) inducer in an otherwise ER-α negative breast tissue, is of great interest. However, the results shown here suggest that additional animal toxicological and bioavailability studies are required to establish a role of GW9662 as a chemopreventive agent.

Pharmacokinetics and tissue and tumor exposure of CP-31398, a p53-stabilizing agent, in rats.

PURPOSE: CP-31398 (N0-[2-[(E)-2-(4-methoxyphenyl)ethenyl] quinazolin-4-yl]-N,N-dimethylpropane-1,3-diamine hydrochloride) is one of the new class of agents that can stabilize the DNA-binding domain of p53 and thereby maintain the activity of p53 as a tumor suppressor and transcription factor. Through its activity as a p53 stabilizer, CP-31398 demonstrates significant cancer preventive and therapeutic activity in several in vivo animal models. The objective of the current study was to describe the pharmacokinetic profile and tissue distribution of this novel agent following intravenous or oral (gavage and dietary) administration. METHODS: CP-31398 was administered to male CD and F344 rats as a single intravenous bolus dose or by daily oral gavage dosing. Male F344 rats also received drug as an ad libitum dietary supplement. Plasma, liver, skin, colon, and colon tumor samples were collected after oral dosing. Concentrations of CP-31398 in plasma and tissue samples were analyzed using LC­MS/MS, and the resultant data were subjected to a non-compartmental pharmacokinetic analysis. RESULTS: Bioavailability (12­32%), elimination half-life (14­20 h), clearance (4.2­4.8 l/h/kg), and volume of distribution (70­82 l/kg) were determined. Tissue levels of CP-31398 after oral (gavage or diet) administration were several orders of magnitude higher than were corresponding plasma concentrations; CP-31398 levels were especially high in colon and liver. Levels of CP-31398 in tissues were higher after gavage dosing than after dietary administration. CONCLUSIONS: CP-31398 is bioavailable and has a relatively long elimination half-life, which supports the achievement of plasma steady-state levels with a once daily dosing regimen. CP-31398 exhibits a dramatically high volume of distribution, which is consistent with its tissue concentrations being much higher than corresponding plasma levels. It is accumulated in colon tumor tissues, albeit at lower concentrations than found in liver, skin, and colon.

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.

Toxicogenomics and metabolomics of pentamethylchromanol (PMCol)-induced hepatotoxicity.

Pentamethyl-6-chromanol (PMCol), a chromanol-type compound related to vitamin E, was proposed as an anticancer agent with activity against androgen-dependent cancers. In repeat dose-toxicity studies in rats and dogs, PMCol caused hepatotoxicity, nephrotoxicity, and hematological effects. The objectives of this study were to determine the mechanisms of the observed toxicity and identify sensitive early markers of target organ injury by integrating classical toxicology, toxicogenomics, and metabolomic approaches. PMCol was administered orally to male Sprague-Dawley rats at 200 and 2000 mg/kg daily for 7 or 28 days. Changes in clinical chemistry included elevated alanine aminotransferase, total bilirubin, cholesterol and triglycerides-indicative of liver toxicity that was confirmed by microscopic findings (periportal hepatocellular hydropic degeneration and cytomegaly) in treated rats. Metabolomic evaluations of liver revealed time- and dose-dependent changes, including depletion of total glutathione and glutathione conjugates, decreased methionine, and increased S-adenosylhomocysteine, cysteine, and cystine. PMCol treatment also decreased cofactor levels, namely, FAD and increased NAD(P)+. Microarray analysis of liver found that differentially expressed genes were enriched in the glutathione and cytochrome P450 pathways by PMCol treatment. Reverse transcription-polymerase chain reaction of six upregulated genes and one downregulated gene confirmed the microarray results. In conclusion, the use of metabolomics and toxicogenomics demonstrates that chronic exposure to high doses of PMCol induces liver damage and dysfunction, probably due to both direct inhibition of glutathione synthesis and modification of drug metabolism pathways. Depletion of glutathione due to PMCol exposure ultimately results in a maladaptive response, increasing the consumption of hepatic dietary antioxidants and resulting in elevated reactive oxygen species levels associated with hepatocellular damage and deficits in liver function.

Determination and stability of CP-31398 in plasma from experimental animals by LC-MS/MS.

A sensitive and accurate approach for the determination of CP-31398 (N-{2-[(E)-2-(4-methoxy-phenyl)-vinyl]-quinazolin-4-yl}-N',N'-dimethyl-propane-1,3-diamine hydrochloride) in rat and dog plasma by LC-MS/MS was validated to support preclinical toxicological and pharmacological studies. Based on the results of stability experiments with diluted CP-31398 solutions using NMR, LC-MS/MS and LC-Q-TOF, all sample preparation and handling steps were performed under yellow light to avoid CP-31398 decomposition. CP-31398 was extracted by protein precipitation with acetonitrile and separated using a Phenomenex Luna 3µm phenyl-hexyl, 100Å, 30×2.0mm column (rat plasma) or a Phenomenex Synergi 4µ Polar-RP, 80Å, 30×2.0mm column (dog plasma) at a flow rate of 0.30mL/min. The mobile phase consisted of A: 1% formic acid in water and B: 1% formic acid in methanol or acetonitrile. Total run times for rat and dog samples were 7 and 8min, respectively, with accompanying retention times of 1.8 for both columns. A turbo ion spray interface was used as the ion source operating in positive mode. Calibration curves were linear from 5 to 1000ng/mL. Linearity was assessed using the external standard method. Within-run and between-run accuracy was 93-109% of the true value for all analytes with precision (SD) of 8% or less for all experiments. The validated method was applied to preclinical toxicology studies in rats and dogs after oral administration of CP-31398.

Subchronic oral toxicity and metabolite profiling of the p53 stabilizing agent, CP-31398, in rats and dogs.

CP-31398 (N'-[2-[2-(4-methoxyphenyl)ethenyl]-4-quinazolinyl]-N,N-dimethyl-1,3-propanediamine dihydrochloride) is a styrylquinazoline that stabilizes the DNA binding conformation of p53, thereby maintaining the activity of p53 as a transcription factor and tumor suppressor. In consideration of the potential use of p53 stabilizers for cancer prevention and therapy, 28-day studies (with recovery) were performed to characterize the toxicity of CP-31398 in rats and dogs. In the rat study, groups of 15 CD rats/sex received daily gavage exposure to CP-31398 at 0, 40, 80, or 160mg/kg/day (0, 240, 480, or 960mg/m(2)/day). In the dog study, groups of five beagle dogs received daily gavage exposure to CP-31398 at 0, 10, 20, or 40mg/kg/day (0, 200, 400, or 800mg/m(2)/day). The high dose of CP-31398 induced mortality in both species: seven male rats and four female rats died as a result of hepatic infarcts, and two female dogs died as a result of hepatic necrosis without evidence of thrombosis. No deaths were seen in the mid- or low-dose groups in either species. In dogs, sporadic emesis was seen in the high dose and mid dose groups, and reductions in body weight gain were observed in all drug-exposed groups. CP-31398 induced mild anemia in both species; clinical pathology data also demonstrated hepatic toxicity, renal toxicity, inflammatory reactions, and coagulopathies in rats in the high dose and mid dose groups. Treatment-related microscopic changes in high dose and mid dose rats were identified in the liver, kidney, heart, bone marrow, lung, adrenals, spleen, thymus, skeletal muscle, and ovary; microscopic changes in the liver, heart, lung, and adrenals persisted through the recovery period. In dogs, microscopic changes were identified in the central nervous system, lung, and liver; changes in all tissues remained at the end of the recovery period. The liver is the primary site of limiting toxicity for CP-31398 in rats, and is also a key site of toxicity in dogs. The maximum tolerated dose (MTD) for subchronic oral administration of CP-31398 is 80mg/kg/day (480mg/m(2)/day) in rats and 20mg/kg/day (400mg/m(2)/day) in dogs. Although only modest and apparently reversible toxicities (microscopic changes in rats; reductions in body weight gain and alterations in red cell parameters in dogs) were seen in the low dose groups, no observed adverse effect levels (NOAELs) for CP-31398 could not be established for either species. The toxicity of CP-31398 suggests that this agent may not be suitable for use in cancer prevention. However, should in vivo antitumor efficacy be achievable at doses that do not induce limiting toxicity, CP-31398 may have utility as a cancer therapeutic. Modification of the primary sites of CP-31398 metabolism (N-demethylation of the alkyl side chain; hydroxylation and O-demethylation of the styryl benzene group) may result in the development of CP-31398 analogs with comparable pharmacologic activity and reduced toxicity.

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.

Pharmacokinetics, oral bioavailability, and metabolic profile of resveratrol and its dimethylether analog, pterostilbene, in rats.

PURPOSE: Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a naturally occurring polyphenol with a broad range of possible health benefits, including anti-cancer activity. However, the biological activity of resveratrol may be limited by poor absorption and first-pass metabolism: only low plasma concentrations of resveratrol are seen following oral administration, and metabolism to glucuronide and sulfate conjugates is rapid. Methylated polyphenol analogs (such as pterostilbene [3,5-dimethoxy-4'-hydroxy-trans-stilbene], the dimethylether analog of resveratrol) may overcome these limitations to pharmacologic efficacy. The present study was designed to compare the bioavailability, pharmacokinetics, and metabolism of resveratrol and pterostilbene following equimolar oral dosing in rats. METHODS: The agents were administered orally via gavage for 14 consecutive days at 50 or 150 mg/kg/day for resveratrol and 56 or 168 mg/kg/day for pterostilbene. Two additional groups were dosed once intravenously with 10 and 11.2 mg/kg for resveratrol and pterostilbene, respectively. Plasma concentrations of agents and metabolites were measured using a high-pressure liquid chromatograph-tandem mass spectrometer system. Noncompartmental analysis was used to derive pharmacokinetic parameters. RESULTS: Resveratrol and pterostilbene were approximately 20 and 80% bioavailable, respectively. Following oral dosing, plasma levels of pterostilbene and pterostilbene sulfate were markedly greater than were plasma levels of resveratrol and resveratrol sulfate. Although plasma levels of resveratrol glucuronide exceeded those of pterostilbene glucuronide, those differences were smaller than those of the parent drugs and sulfate metabolites. CONCLUSIONS: When administered orally, pterostilbene demonstrates greater bioavailability and total plasma levels of both the parent compound and metabolites than does resveratrol. These differences in agent pharmacokinetics suggest that the in vivo biological activity of equimolar doses of pterostilbene may be greater than that of resveratrol.

Improved oral bioavailability in rats of SR13668, a novel anti-cancer agent.

PURPOSE: SR13668, a bis-indole with potent activity in vitro and in vivo against various cancers and promising cancer chemopreventive activity, was found to have very low oral bioavailability, <1%, in rats during pilot pharmacokinetic studies. The objective of these studies was to better understand the source of low oral exposure and to develop a formulation that could be used in preclinical development studies. METHODS: An automated screening system for determining solubility in lipid-based vehicles, singly and in combination, was used to identify formulations that might enhance absorption by improving solubility of SR13668, and these results were confirmed in vivo using Sprague-Dawley rats. Pharmacokinetics of SR13668 was then determined in male and female Sprague-Dawley rats administered 1 mg/kg iv, 1, 10, and 30 mg/kg po formulated in PEG400:Labrasol (1:1 v/v). Blood was collected at time points through 24 h and the concentration of SR13668 determined using HPLC with UV and fluorescence detection. RESULTS: SR13668 was found to be resistant to plasma esterases in vitro and relatively stable to rat and human liver microsomal metabolism. SR13668 concentrates in tissues as indicated by significantly higher levels in lung compared to blood, blood concentrations ~2.5-fold higher than plasma levels, and apparent volume of distribution (V) of ~5 l/kg. A marked sex difference was observed in exposure to SR13668 with area under the curve (AUC) significantly higher and clearance (CL) lower for female compared to male rats, after both iv and oral administration. The oral bioavailability (F) of SR13668 was 25.4 ± 3.8 and 27.7 ± 3.9% (30 mg/kg), for males and females, respectively. A putative metabolite (M1), molecular weight of 445 in the negative ion mode (i.e., SR13668 + 16), was identified in blood samples from both the iv and po routes, as well as in vitro microsomal samples. CONCLUSIONS: In summary, while SR13668 does undergo metabolism, probably by the liver, the oral bioavailability of SR13668 in rats was dramatically improved by the use of formulation that contained permeation enhancers and promoted better solubilization of the drug.

Assessment of oral toxicity and safety of pentamethylchromanol (PMCol), a potential chemopreventative agent, in rats and dogs.

2,2,5,7,8-Pentamethyl-6-chromanol (PMCol) was administered by gavage in rats for 28 days at dose levels of 0, 100, 500, and 2000mg/kg/day. PMCol administration induced decreases in body weight gains and food consumption, hepatotoxicity (increased TBILI, ALB, ALT, TP; increased relative liver weights; increased T4 and TSH), nephrotoxicity (increased BUN and BUN/CREAT, histopathology lesions), effect on lipid metabolism (increased CHOL), anemia, increase in WBC counts (total and differential), coagulation (FBGN upward arrow and PT downward arrow) and hyperkeratosis of the nonglandular stomach in the 2000mg/kg/day dose group (in one or both sexes). In the 500mg/kg/day dose group, toxicity was seen to a lesser extent. In the 100mg/kg/day dose group, only increased CHOL (females) was observed. To assess the toxicity of PMCol in male dogs it was administered orally by capsule administration for 28 days at dose levels of 0, 50, 200 and 800mg/kg/day (four male dogs/dose group). PMCol treatment at 800mg/kg/day resulted in pronounced toxicity to the male dogs. Target organs of toxicity were liver and thymus. Treatment at 200mg/kg/day resulted in toxicity consistent with slight adverse effect on the liver only. The results of the safety pharmacology study indicate that doses of 0, 50, 200 and 800mg/kg administered orally did not have an effect on the QT interval, blood pressures and body temperatures following dosing over a 24-h recording period. Under the conditions of this study, the no-observed-adverse effect level (NOAEL) for daily oral administration of PMCol by gavage for 28 days to male rats was 100mg/kg/day and 50mg/kg in male dogs. In female rats, the NOAEL was not established due to statistically significant and biologically meaningful increases in CHOL level seen in the 100mg/kg/day dose group. The results of these studies indicated that administration of PMCol at higher dose levels resulted in severe toxicity in dogs and moderate toxicity in rats, however, administration at lower levels is considered to be less likely to result in toxicity following 28 days of exposure. Sex-related differences were seen in rats. Male rats appeared to have greater sensitivity to nephrotoxicity, while female animals had a greater incidence of hepatoxicity and changes in hematological parameters evaluated, especially at a dose of 500mg/kg/day, which correlated to the higher plasma drug levels in female rats. It appeared that dogs were generally more sensitive than rats to oral administration of PMCol. Further examination of the potential toxic effects of PMCol in longer term studies is required prior to understanding the full risks of PMCol administration as a chemopreventative agent.

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.

Pharmacokinetic and tissue distribution study of [14C]fluasterone in male Beagle dogs following intravenous, oral and subcutaneous dosing routes.

The purpose of this work was to compare the pharmacokinetics (PK) and tissue distribution of [14C]fluasterone following intravenous (iv), subcutaneous (sc) and oral (po) administration in male Beagle dogs. The main goal of the investigation was to discover if non-oral routes would alter parameters observed in this study following the administration of [14C]fluasterone. The oral formulation had a lower bioavailability (47%) compared to the sc formulation (84%). Po and sc administration resulted in a similar t(max); however, the observed C(max) following sc dosing was less than half of that after oral dosing. The sc route had the greatest overall exposure (AUC(0-infinity)). Tissue distribution analysis 2 h post-intravenous dosing showed that connective tissue (adipose and bone), liver, and skeletal muscle accumulated relatively high levels of fluasterone. The majority of the dose was retained during the first 24 h. Elimination of [14C]fluasterone-derived radioactivity following intravenous dosing resulted in urine and feces containing 7.6% and 28%, respectively, of the total dose over the first 24 h. Elimination of [14C]fluasterone-derived radioactivity following subcutaneous dosing resulted in 4.6% in urine and 7.8% in feces of the total dose over the first 24 h. Following oral dosing, elimination resulted in 3.8% in urine and 36% in feces over the first 24h. In conclusion, the sc route of administration offers some advantages to po and iv due to the prolonged release and increased retention through 24 h.

Pharmacokinetics and enhanced bioavailability of candidate cancer preventative agent, SR13668 in dogs and monkeys.

PURPOSE: SR13668 (2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo-(2,3-b)carbazole), is a new candidate cancer chemopreventive agent under development. It was designed using computational modeling based on a naturally occurring indole-3-carbinol and its in vivo condensation products. It showed promising anti-cancer activity and its preclinical toxicology profile (genotoxicity battery and subchronic rat and dog studies) was unremarkable. However, it exhibited a very poor oral bioavailability (<1%) in both rats and dogs. Therefore, a study was initiated to develop and evaluate in dogs and non-human primates formulations with a more favorable oral bioavailability. METHODS: Two formulations utilizing surfactant/emulsifiers, PEG400:Labrasol and Solutol, were tested in dogs and monkeys. Levels of SR13668 were measured in plasma and blood using a high-performance liquid chromatograph-tandem mass spectrometer system. Non-compartmental analysis was used to derive pharmacokinetic parameters including the bioavailability. RESULTS: The Solutol formulation yielded better bioavailability reaching a maximum of about 14.6 and 7.3% in dogs and monkeys, respectively, following nominal oral dose of ca. 90 mg SR13668/m(2). Blood levels of SR13668 were consistently about threefold higher than those in plasma in both species. SR13668 did not cause untoward hematology, clinical chemistry, or coagulation effects in dogs or monkeys with the exception of a modest, reversible increase in liver function enzymes in monkeys. CONCLUSIONS: The lipid-based surfactant/emulsifiers, especially Solutol, markedly enhanced the oral bioavailability of SR13668 over that previously seen in preclinical studies. These formulations are being evaluated in a Phase 0 clinical study prior to further clinical development of this drug.

In-vitro and in-vivo metabolic studies of the candidate chemopreventative pentamethylchromanol using liquid chromatography/tandem mass spectrometry.

OBJECTIVES: This study focuses on the in-vitro metabolic profiles of pentamethylchromanol in human, rat, dog and non-human primates, and characterizes the associated metabolic kinetics and specific human isozymes responsible for metabolism. Additional investigations compare in-vitro data with in-vivo metabolic data from rats and dogs. METHODS: In-vitro metabolites were generated from commercially available microsomes, S9 fractions and cytochrome P450 isozymes. Reaction mixtures were analysed using liquid chromatography/tandem mass spectrometry for metabolite identification, stability, phenotyping and kinetic profiles. Plasma samples were collected from 28-day toxicology studies in rats and dogs, and analysed using the same methodology as for the identification of in-vitro metabolites. KEY FINDINGS: Samples from in-vitro experiments produced a total of eight identified metabolites while five were observed in the in-vivo samples. Kinetic analysis of metabolites in human microsomes generated Michaelis constants (K(M)) ranging from 10.9 to 104.9 mum. Pentamethylchromanol metabolic stability varied by species and multiple isozymes were identified for the observed biotransformation pathways. Pentamethylchromanol is susceptible to multiple metabolic pathways and differential metabolic stability, which is species dependent. CONCLUSIONS: In-vitro metabolism was not a strong predictor of in-vivo metabolism for the samples assays but showed glucuronidation and sulfation as common biotransformation pathways.

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.