Hr Postdosing Research Articles

PHARMACOKINETICS OF MELOXICAM FOLLOWING A SINGLE ORAL DOSE IN MALAYAN FLYING FOXES ( PTEROPUS VAMPYRUS).

Meloxicam, a COX-2 selective nonsteroidal anti-inflammatory medication, has been used in many exotic animals at doses extrapolated from domestic animal pharmacokinetic and pharmacodynamic studies. Increasing evidence suggests that significant species differences exist in meloxicam metabolism. Because of this, dose extrapolation from domestic animals may not be appropriate for exotic species. The objective of this study was to investigate the pharmacokinetics of meloxicam in a population of male Malayan flying foxes, Pteropus vampyrus, following a single oral dose of 0.2 mg/kg. Using a sparse sampling method based on a pilot study, two blood samples from each of 10 bats were collected over an 8-hr time period. Analysis of meloxicam in plasma samples was conducted using reversed-phase high-performance liquid chromatography. The peak plasma concentration of 598 ± 157.5 ng/ml occurred at 1.0 hr post dosing. The terminal half-life was 1.1 ± 0.1 hr, which indicates that meloxicam is rapidly metabolized in this species. No adverse clinical effects were noted during the study period. A single oral dose of 0.2 mg/kg appears safe for use in male Malayan flying foxes, but due to rapid elimination, frequent dosing may be required to maintain plasma concentrations within a therapeutic range. Multidose studies are needed to determine if plasma accumulation of meloxicam occurs.

Acute Alpha-Naphthylisothiocyanate-induced Liver Toxicity in Germfree and Conventional Male Rats.

Differences in the responses of conventional and germfree male Sprague-Dawley rats to acute injury induced by alpha-naphthylisothiocyanate (ANIT), a well-characterized biliary epithelial toxicant, were evaluated. Conventional and germfree rats were dosed once orally with 50 mg/kg of ANIT or corn oil alone and serially sacrificed daily for the next 3 days. Germfree rats treated with ANIT tended to have greater increases in virtually all liver and biliary-related analytes compared with conventional rats treated with ANIT; however, significant differences were found only in a few of these analytes including increased bile acids on day 3, total bilirubin on day 4, glutamate dehydrogenase (GLDH) on day 3, and reduced paraoxonase 1 (PON1) on days 2 and 3. Histologic differences between the conventional and germfree rats were modest, but most pronounced on day 2 (24-hr post dosing). Based on subjective scoring, biliary necrosis, neutrophilic cholangitis, and portal tract edema were more severe in germfree rats at 24 hr post dosing compared with conventional rats. Biliary epithelial replication did not differ between treated groups, however. Overall, germfree rats had a modestly greater level of biliary tract injury based on subjective histologic scoring and clinical chemistry measurements following an acute exposure to the well-characterized biliary toxin, ANIT; however, the difference between the ANIT-treated germfree and conventional groups was modest and most evident only within the first day following exposure. These findings suggest that the microbiome did not significantly affect ANIT-induced acute biliary tract injury in the conditions of this study.

Abstract 3121: Evaluation of novel combinations of PI3K-mTOR inhibitors with dacomitinib (dac) or chemotherapy in PTEN-deficient genomically characterized patient-derived tumor xenografts (gPTX)

Abstract Background: Activation of the PI3K pathway has been associated with resistance to anti-human epidermal growth factor receptor (HER) therapy providing a rationale to combine PI3K-mTOR inhibitors and the pan-HER inhibitor dac. Loss of PTEN function may be associated with sensitivity to PI3K-mTOR inhibitors. The objectives of these experiments are to evaluate the combination of PI3K-mTOR inhibitors with chemotherapy or dac in PTEN-deficient gPTX models. Material and Methods: Three gPTX models [triple-negative breast cancer (TNBC), low-grade serous ovarian cancer (LGSOC), and non-small cell lung cancer (NSCLC)] were selected based on their deficient expression of PTEN by immunohistochemistry. Two dual PI3K-mTOR inhibitors were evaluated: PF-04691502 (5 mg/kg, daily, oral gavage) and PF-05212384 (10 mg/kg, twice weekly, intravenously). Three different combinations were evaluated: PI3K-mTOR inhibitor with 1) cisplatin (3 mg/kg, once weekly, intraperitoneally -IP); 2) with paclitaxel (10 mg/kg, twice weekly, IP); 3) with dac (3 mg/kg daily, oral gavage). This last experiment contained an acute dose component to evaluate target modulation by collecting tumor samples at 1 hr and 24 hr post dosing. Results: The percentages of tumor growth inhibition (TGI%) induced by the different treatment arms and daily tumor volume change (ΔV) for each arm compared to the control arm (p-value) are summarized in Table 1. Pharmacodynamic modulation of pS6 and pAKT was observed in the acute dose experiment in the arms of PI3K-mTOR inhibitor with or without dac. Conclusions: In gPTX with PTEN loss, the addition of a PI3K-mTOR inhibitor may improve the TGI% when compared to chemotherapy or dac alone. This benefit was largely offset in the NSCLC gPTX harboring KRAS G12C and/or TP53 mutations. The role of PTEN deficiency in the antitumor activity of these combinations should be further investigated in the clinic. Table 1gPTX tumor typeTNBCLGSOCNSCLCMolecular profilePTEN nullKRAS G12RPTEN lowKRAS G12CTP53 R181PPTEN lowTGI%ΔVp-valueTGI%ΔVp-valueTGI%ΔVp-valueExperiment 1PF-0521238432%0.0739%0.19-1%0.78Cis42%<0.05**43%0.0397%<0.05**PF-05212384 + Cis96%<0.05**68%<0.05**92%<0.05**Experiment 2PF-0521238422%0.6440%0.21PF-0469150233%<0.05**Pac84%<0.05**38%0.2079%<0.05**PF-05212384 + Pac110%^<0.05**56%<0.05**PF-04691502 + Pac45%<0.05**Experiment 3PF-0521238422%0.6140%0.26PF-0469150233%<0.05**Dac15%0.998%0.5323%0.35PF-05212384 + Dac55%0.0945%0.47PF-04691502 + Dac32%0.055Cis = cisplatin; Pac = paclitaxel; Dac = dacomitinib; TNBC = triple negative breast cancer; LGSOC = low grade serous ovarian cancer; NSCLC = non-small cell lung cancerTGI%: percentages of tumor growth inhibition; ΔV: differences between daily tumor volume change of each treatment arm and the control arm^ Tumor regression; ** statistically significant Citation Format: Irene Brana, Nhu-An Pham, Lucia Kim, Shingo Sakashita, Ming Li, Christine Ng, Yuhui Wang, Peter Loparco, Jose Rafael Sierra, Lisa Wang, Lillian L. Siu, Ming S. Tsao. Evaluation of novel combinations of PI3K-mTOR inhibitors with dacomitinib (dac) or chemotherapy in PTEN-deficient genomically characterized patient-derived tumor xenografts (gPTX). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3121. doi:10.1158/1538-7445.AM2014-3121

Cardiac Troponin I in Isoproterenol-Induced Cardiac Injury in the Hanover Wistar Rat: Studies on Low Dose Levels and Routes of Administration

The current studies demonstrate the effect of low-dose intraperitoneal (IP) administration of isoprotenerol (ISO) and subcutaneous (SC) versus IP routes of administration of ISO on serum cardiac troponin I (cTnI) levels in female Hanover Wistar rats, providing additional evidence to support acceptance of cTnI as a cardiac biomarker. At 2 hr postdosing with 0-500 microg/kg ISO, mean serum cTnI levels were increased in a dose-related fashion at > or =10 microg/kg with no evidence of cardiac pathology. At 24 h, cTnI concentrations were generally at control levels, but histologic cardiomyocyte injury was evident in a proportion of the animals given > or =10 microg/kg. In a second experiment, rats given SC ISO at 5,000 microg/kg and necropsied at 0, 1, 2, and 4 hr postdosing had higher levels of serum cTnI than animals given the same dose IP.

The pharmacokinetics of azacitidine following subcutaneous treatment in patients with myelodysplastic syndromes (MDS) or acute myelogenous leukemia (AML)

7087 Background: 5-azacitidine (AZA), through its effects on DNA metabolism, gene expression, and cell differentiation, has proven beneficial in treatment of MDS and AML and AZA therapy significantly increases survival in higher-risk MDS and AML compared to conventional care. Few studies have evaluated the pharmacokinetics (PK) of AZA and the renal elimination of AZA has not been previously published to our knowledge. Plasma PK of AZA are herein reported in patients receiving SC doses of 75 mg/m2. This study was designed to also assess the contribution of renal elimination to the overall clearance of AZA. Methods: Adult patients with MDS or AML and ECOG status 0–2 were treated with 7 consecutive daily SC doses of 75 mg/m2 AZA during their first treatment cycle. PK parameters of AZA were derived from drug concentrations in plasma and urine collected after the first and last dose (day 7) of AZA. Safety was evaluated by adverse event reporting (NCI-CTC). Results: Currently, 18 patients have been treated with SC AZA. AZA was rapidly absorbed and reached peak plasma concentrations (concs) within 0.5 hr post dosing. The AUCinf after SC doses was 1170 hr*ng/mL. The AZA concs declined in a pseudo bi-phasic manner with an elimination half-life of 1.25 hours. The plasma PK profiles after the first and last dose were superimposable. The apparent total clearance (CL/F) and volume of distribution (Vd/F) were 143 L/hr and 318 L, respectively. AZA recovery in urine was very small relative to dose (<2%). AZA was well tolerated and no unexpected toxicities were observed. Conclusions: The AZA AUCinf after SC doses is similar to the published AUC value (1044 hr*ng/mL) after 75 mg/m2 IV doses indicating approximating 100% systemic bioavailability. After SC dosing, CL/F exceeded hepatic blood flow indicating extra-hepatic metabolism. Vd/F was 4–5 fold greater than total body water suggesting extensive AZA tissue distribution. Renal elimination appears to play a minor role in the overall clearance of AZA. [Table: see text]

Validation and use of three complementary analytical methods (LC–FLS, LC–MS/MS and ICP–MS) to evaluate the pharmacokinetics, biodistribution and stability of motexafin gadolinium in plasma and tissues

Liquid chromatography-fluorescence (LC-FLS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and inductively coupled plasma-mass spectrometry (ICP-MS) methods were developed and validated for the evaluation of motexafin gadolinium (MGd, Xcytrin) pharmacokinetics and biodistribution in plasma and tissues. The LC-FLS method exhibited the greatest sensitivity (0.0057 microg mL(-1)), and was used for pharmacokinetic, biodistribution, and protein binding studies with small sample sizes or low MGd concentrations. The LC-MS/MS method, which exhibited a short run time and excellent selectivity, was used for routine clinical plasma sample analysis. The ICP-MS method, which measured total Gd, was used in conjunction with LC methods to assess MGd stability in plasma. All three methods were validated using human plasma. The LC-FLS method was also validated using plasma, liver and kidneys from mice and rats. All three methods were shown to be accurate, precise and robust for each matrix validated. For three mice, the mean (standard deviation) concentration of MGd in plasma/tissues taken 5 hr after dosing with 23 mg kg(-1) MGd was determined by LC-FLS as follows: plasma (0.025+/-0.002 microg mL(-1)), liver (2.89+/-0.45 microg g(-1)), and kidney (6.09+/-1.05 microg g(-1)). Plasma samples from a subset of patients with brain metastases from extracranial tumors were analyzed using both LC-MS/MS and ICP-MS methods. For a representative patient, > or = 90% of the total Gd in plasma was accounted for as MGd over the first hour post dosing. By 24 hr post dosing, 63% of total Gd was accounted for as MGd, indicating some metabolism of MGd.

Plasma levels of 5-hydroxyindole-3-acetic acid (5HIAA) as a pharmacodynamic marker of blood flow changes induced by the vascular targeting agent (VTA) 5,6 dimethyl xanthenone acetic acid, DMXAA

3123 Background: VTAs now undergoing clinical trials include those that interact with tubulin (e.g. combretastatins and ZD6126) and the distinct tubulin-independent based flavonoid DMXAA (AS1404). DMXAA induces both direct apoptosis of tumor vascular endothelial cells and a secondary induction of vasoactive agents such as serotonin (from platelets aggregating to damaged vasculature) and tumor necrosis factor (TNFα). The study objective was to determine whether plasma levels of the serotonin metabolite, 5HIAA, correlated with DMXAA induced blood flow changes, first in mouse models and then in patients. Methods: 5HIAA levels from blood, were determined by HPLC. Mice, bearing syngeneic colon 38 subcutaneous tumors, were given single doses of DMXAA (up to ∼150mg/m2). Within a recently completed Phase I double-blind randomised study in refractory tumors; DART) patients received 20 minute intravenous infusions of DMXAA at 300 to 3000 mg/m2. Results: 5HIAA levels in mice measured 4hr post DMXAA showed a significant linear correlation with increased extravasation of the albumin binding Evans Blue from tumors (r=0.82; P<0.05); extravasation significantly correlated with reduced tumor blood flow (r = 0.88; P<0.01). In the same mice, no change in extravasation was seen in normal skin. In patients, peak 5HIAA plasma levels occurred at 4hr post dosing at dose levels >600mg/m2. Notably, there was a positive correlation between 5HIAA plasma levels and DMXAA dose up to 1200mg/m2 but thereafter a plateau was observed (even though plasma levels of free DMXAA increased linearly with dose up to 3000mg/m2). Conclusions: Increased plasma levels of 5HIAA may represent a sensitive biological marker of blood flow changes induced by the VTA, DMXAA. Dose-response data from Phase I trial patients receiving DMXAA show that the optimum biological dose to cause tumor blood flow/5HIAA changes is in the range of 1200mg/m2, that is, well below the maximum tolerated dose. Consequently, doses in this range are being studied in Phase II combination trials with taxanes (where marked synergy was seen in various preclinical tumor models) and platins. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Antisoma Antisoma

Embryo/fetal toxicity assessment of lasofoxifene, a selective estrogen receptor modulator (SERM), in rats and rabbits.

The purpose of this study was to evaluate the effects of lasofoxifene, a selective estrogen receptor modulator (SERM), on rat and rabbit fetal development. Lasofoxifene was administered orally to rats (1, 10, 100 mg/kg) between gestation days (GD) 6-17, and in rabbits (0.1, 1, 3 mg/kg) between GD 6-18. Maternal body weight and food consumption were monitored throughout pregnancy. Fetuses were delivered by Cesarean section on GD 21 in rats, and GD 28 in rabbits, to evaluate fetal viability, weight, and morphology. Drug concentrations in maternal plasma were measured in a separate cohort of animals at several time points commencing on GD 17 (rats) and 18 (rabbits). On GD 18 (rat) and GD 19 (rabbit) drug concentrations were measured in maternal plasma and in fetal tissue 2 hr post dosing to determine the fetal to maternal drug ratio. In rats, there were dose-related declines in maternal weight gain and food consumption. Post implantation loss was significantly increased at dosages of 10 and 100 mg/kg, and the number of viable fetuses was decreased at 100 mg/kg. The placental weights increased, whereas fetal weights decreased in a dose-dependent manner. Lasofoxifene-related teratologic findings were noted at 10 and 100 mg/kg and included imperforate anus with hypoplastic tails, dilatation of the ureters and renal pelvis, misaligned sternebrae, hypoflexion of hindpaw, wavy ribs, and absent ossification of sternebrae. In rabbits, neither maternal weight gain nor food consumption were affected during treatment. Between GD 26-28, there was a dose-dependent increased incidence of red discharge beneath the cages. At 1 and 3 mg/kg, resorptions and post-implantation loss increased. There were no significant external or visceral effects, but 3 mg/kg there was an increased incidence of supernumerary ribs. Although the maternal plasma Cmax and AUC(0-24) were dose-dependent, the exposures in the rat were many orders of magnitude greater than in the rabbit even for the same 1 mg/kg dose. The single time point fetal/maternal drug ratio was higher in the rat (1.3-0.78) than in the rabbit (0.21-0.16). In general, both maternal and fetal effects of lasofoxifene were similar to those reported with other SERMs. Although the incidence or severity of these effects was, in some instances, greater in the rat than in the rabbit, the doses and the resultant maternal and fetal exposures were many orders of magnitude higher in the rat, suggesting the rabbit to be more sensitive to the toxicological effects of lasofoxifene.

Comparison of Injectable Versus Oral Enrofloxacin Pharmacokinetics in Red-Eared Slider Thirties, Trachemys scripta elegans

ABSTRACT Three male and three female red-eared slider turtles, Trachemys scripta elegans, were given an intramuscular (IM) injection of enrofloxacin (5 mg/kg body weight). Blood was obtained from a peripheral vein, and plasma concentrations of enrofloxacin and ciprofloxacin were measured at 0, 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 hr post injection. After a period of six weeks, the same animals were given oral doses of enrofloxacin (10 mg/kg body weight). Blood was obtained and plasma concentrations of enrofloxacin and ciprofloxacin were measured at 1, 2, 5, 8, 12, 24, 36, 48, 72 hr post oral dosing. The highest mean concentrations of enrofloxacin and ciprofloxacin were 6.28 μg/ml 2 hr and 0.42 μg/ml 7.4 hr after IM injection and 3.44 μg/ml 5 hr and 0.35 μg/ml 6 hr after oral administration. The mean apparent elimination half-life of enrofloxacin was 17.6 hr after IM injection and 32.8 hr after oral administration. The half-life for ciprofloxacin was 28.4 hr post IM administration and 59.6 hr post-oral admi...

Systemic efficacy of nodulisporic acid against fleas on dogs.

Nodulisporic acid A (NSA) is a novel natural product from a new structural class that was shown previously to have insecticidal activity against blowfly larvae. To determine if there was useful systemic efficacy against fleas (Ctenocephalides felis). NSA was evaluated in an artificial membrane flea feeding device and in dogs. In the artificial membrane flea feeding device, adult C. felis were allowed to feed on bovine blood containing various concentrations of NSA through a Parafilm membrane. NSA killed the fleas with a 50% lethal concentration of 0.68 microg/ml and was approximately 10-fold more potent than the systemic insecticide ivermectin. In the initial probe dog test, a single beagle was challenged with 100 C. felis before oral dosing with 15 mg/kg of NSA. Flea counts conducted at 72 hr postdosing showed an 88% reduction relative to control. Re-challenge of the same dog at 5 days postdosing showed 50% reduction of fleas at day 7, demonstrating some residual flea activity. In a confirmatory study, 8 dogs were challenged with 100 fleas just before oral dosing with 15 mg/kg of NSA (4 dogs) or vehicle (4 dogs). There was 99% reduction of fleas at 48 hr postdosing in the NSA-treated dogs relative to control. Additional challenges with 100 fleas were performed on these 8 dogs at 48-hr intervals to determine the duration of efficacy, and there was 97, 51, and 0% reduction of fleas relative to control on days 4, 6, and 8, respectively. No adverse effects were observed in the dogs in these studies. These data show that NSA has potent oral activity in the dog for the control of fleas, while lacking overt mammalian toxicity.

Absorption, Distribution and Excretion of OPB-2045 following a Single Subcutaneous Administration to Rats.

Examination was made of the absorption, distribution and excretion of radioactivity in SpragueDawly rats following a single subcutaneous administration of 14C-labeled OPB-2045 at 1 mg/kg, and the following results were obtained: 1. Serum concentrations of radioactivity were maximum (Cmax) of 0.032-0.033 μg eq./ml at 1 hr after administration in male and female rats. Radioactivity half-life in serum was 77.0 hr as determined at 48 hr to 168 hr postdosing in male and female rats. The area under the serum concentration-time curve (AUC0→∞) was 0.75 to 0.78 μg eq.·hr/ml. Radioactivity in blood cells accounted for 31.9-77.9% of total radioactivity in the blood. Radioactivity in the blood exceeded that in the serum. AUC0→∞ in the blood was 1.5 to 2 times that of the serum. 2. Radioactivity was highest in the pituitary gland, followed by the thyroid, adrenal glands, lungs and kidneys in male rats 1 hr postdosing. In tissues, it was 10 to 26 times higher than in the blood. At 8 and 24 hr postdosing, radioactivity in adrenal glands was extremely high, and 100 to 200 times that in the blood. At 72 and 168 hr postdosing, radioactivity was high in the adrenal and pituitary glands and kidneys. Its tissue distribution in female rats was essentially the same as in male rats. 3. Urinary and fecal excretion of radioactivity was 17.2-20.8% and 68.4-71.2% of the administered doses, respectively, with 9.1-9.3% remaining in the carcass at 168 hr postdosing. 4. Biliary and urinary excretion of radioactivity at 24 hr postdosing accounted for 34.1% and 14.2% the administered doses, respectively, in male rats whose bile ducts had been cannulated.

Metabolism of a New Bactericidal Antiseptic, OPB-2045, in Rats following Subcutaneous Administration.

To investigate the metabolism and disposition of OPB-2045, 1-(3, 4-dichlorobenzyl)-5-octylbiguanide, 14C-labeled OPB-2045 was subcutaneously injected to male Sprague-Dawley rats at a dose of 1 mg/kg and the resulting radioactivity in the urine and bile was analyzed using a high-performance liquid radiochromatography. The radioactivity retained at the injection site was also analyzed. 1. The metabolites excreted in the urine included 6-[5-(3, 4-dichlorobenzyl)-1-biguanidino] hexanoic acid (DM-210), 4-[5-(3, 4-dichlorobenzyl)-1-biguanidino] butanoic acid (DM-212), 5-[5-(3, 4-dichlorobenzyl)-1-biguanidino] pentanoic acid (DM-213) and 3, 4-dichlorobenzoic acid. Within 72 hr after the dosing, the metabolites DM-210, DM-212, DM-213, and 3, 4-dichlorobenzoic acid excreted in the urine accounted for 4.6, 1.6, 1.5 and 1.4% of the administered dose, respectively. No excretion of the unchanged compound was detected in the urine. 2. The metabolites excreted in the bile included DM-210, DM-212 and DM-213. The combined excretion rate of these metabolites accounted for 15.6% of the dose within 24 hr postdosing. No excretion of 3, 4-dichlorobenzoic acid or the unchanged OPB-2045 was detected in the bile. 3. The radioactivity retained at the injection site at 1, 8 and 24 hr postdosing accounted for 51.5%, 34.6% and 13.0% of the administered dose, respectively. The unchanged compound was found to account for at least 80.8% of the radioactivity, and no metabolites including DM-210, DM-212, DM-213 and 3, 4-dichlorobenzoic acid, were detected. These results indicated that OPB-2045 was not metabolized by the skin/tissues at the injection site, but instead by other organ(s).

Distribution of tritiated dihydromicrocystin in swine

The distribution of tritiated dihydromicrocystin i [ 3H]2H-MCLR was studied in anesthetized specific-pathogen-free pig. Two doses were administered i.m. and one dose was given via an isolated ileal loop. At 4 hr after i.v. administration of the toxin at 25 μg/kg, 64.6% of the total dose (% TD) was located in the liver, with small amounts distributed to the kidneys (1.2% TD), lungs (1.75% TD), heart (0.22% TD), ileum (0.13% TD) and spleen (0.04% TD). A similar distribution was found at 4 hr postdosing in pigs given 75 μg/kg, although the liver contained a lower fraction of the total dose, at 46.99% TD, and the kidneys had somewhat more, at 2.19% TD, than the low dose. At the high dose, the fractions of the amount given accounted for by the lungs (0.55% TD), heart (0.23% TD), ileum (0.20% TD) and spleen (0.07% TD) were similar to those at the low dose. The livers of the pigs given 75 μg/kg via the ileal loop, at 5 hr postdosing, contained 49.5% TD and the ileum had 33.94% TD. Smaller amounts were distributed to kidneys (1.04% TD), lungs (0.65% TD), heart (0.81% TD) and spleen (0.16% TD). The livers of both groups dose at 75 μg/kg contained higher concentrations of toxin, but lower percentages of the total dose, than the livers of pigs dosed at 25 μg/kg. Larger increases in serum arginase in the two 75 μg/kg groups were associated with histological evidence of more severe liver damage than at the 25 μg/kg dose. Analysis of radiolabelled compounds from hepatic tissue using fast atom bombardment mass spectrometry determined that the primary constituent was [ 3H]2H-MCLR, but two minor radioactive components were also isolated. These findings indicate that [ 3H]2H-MCLR is rapidly concentrated in the liver of swine, whether given i.v. or via an isolated ileal loop, that at extremely toxic doses uptake is slowed, and that it is as toxicologically active as the parent compound.

Pharmacokinetics of Triclopyr (3,5,6-Trichloro-2-pyridinyloxyacetic Acid) in the Beagle Dog and Rhesus Monkey: Perspective on the Reduced Capacity of Dogs to Excrete This Organic Acid Relative to the Rat, Monkey, and Human

The pharmacokinetics of triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid) were measured in the beagle dog and rhesus monkey and compared with the kinetics observed in rats and humans. In addition, studies were conducted in anesthetized dogs to better understand the mechanism by which [14C]triclopyr is eliminated in this species. Triclopyr was dissolved in distilled water, and administered as a single oral dose of 0.5, 5, or 20 mg/kg to three male dogs. A single male rhesus monkey was given an intravenous dose of 30 mg [14C]triclopyr/kg body wt on two occasions separated by 10 days. Anesthetized male dogs, were implanted with venous, arterial, and urethral catheters and given increasing amounts of triclopyr to produce plasma triclopyr levels ranging from 0.3 to 27 μg eq/mL. In the monkey, triclopyr was rapidly eliminated from the plasma (t12 = 6.3 hr) with >95% of the urinary14C activity excreted within 24 hr postdosing. In the dog, orally administered triclopyr was rapidly and effectively absorbed at every dose level with virtually all of it excreted in the urine by 72 hr postdosing. However, the kinetics were slightly nonlinear, and the fraction of the dose excreted in the urine decreased with increasing dose. Several nonlinear processes may collectively contribute to the modest nonlinear pharmacokinetics in the dog. Plasma protein binding of triclopyr in the dog ranged from 94 to 99%, was nonlinear, and was an important determinant in the renal clearance of triclopyr. The nonlinear plasma protein binding indicates that glomerular filtration became disproportionately more important as plasma triclopyr concentration increased. There was good evidence for a high-affinity low-capacity active-secretory process that was saturated by low plasma triclopyr concentrations. As plasma triclopyr concentrations increased, tubular reabsorption begins to exceed secretion, resulting in decreased renal clearance. The volume of distribution, normalized for body weight, was constant across all species. While clearance and half-life could be allometrically scaled to body weight for the rat, monkey, and human, the dog had a much slower clearance and longer half-life for triclopyr elimination than predicted allometrically. These data demonstrate that the pharmacokinetics of triclopyr in the dog are markedly different than in rat, monkey, and human.

Tissue distribution of the cytofectin component of a plasmid-DNA/cationic lipid complex following intravenous administration in mice.

Allovectin-7 is a gene therapy agent that consists of plasmid DNA (pDNA) encoding the human HLA-B7 class I and beta2-microglobulin genes (VCL-1005), complexed with the cationic lipid DMRIE Br and DOPE. A tritiated version of the cytofectin component, DMRIE Br, was synthesized by regiospecific isotope incorporation to a very high specific activity. The 3H-labeled DMRIE/DOPE mixture was complexed with VCL-1005 to produce a radiolabeled version of Allovectin-7. The VCL-1005/3H-DMRIE/DOPE complex was administered intravenously to mice, and the tissue distribution of radioactivity was analyzed 24 hr later. Excretion of radioisotope was monitored for 96 hr post dosing. At 24 hr post administration, a tissue distribution for the radioisotope of liver >> spleen > lung >> heart > brain approximately muscle approximately blood was observed. During the 96-hr period post dose, very little administered radioactivity (<17%) was excreted and the majority of the isotope (83%) remained in the animal. This is the first report on the biodistribution of the cytofectin component of a pDNA-cationic lipid complex for which the distribution of the plasmid component has also been reported.

Effect of Emulphor, an emulsifier, on the pharmacokinetics and hepatotoxicity of oral carbon tetrachloride in the rat.

Emulphor, a polyethoxylated vegetable oil, is now being used widely to incorporate volatile organic compounds (VOCs) and other lipophilic compounds into aqueous solutions for biochemical, pharmacokinetic, and toxicological studies. Previous work in this laboratory demonstrated that 0.25% Emulphor did not alter the kinetics or hepatotoxicity of low doses of CCl4 compared to when the halocarbon was given to rats orally in water. The present study was undertaken as there was concern that higher concentrations of Emulphor (necessary to maintain lipophilic VOCs in stable aqueous emulsions for extended periods) might alter the VOCs' absorption, disposition, and/or toxicity. Dosages of 10 and 180 mg CCl4/kg bw were given, as an aqueous emulsion using 1, 2.5, 5, or 10% Emulphor, by gavage to fasted male Sprague-Dawley rats. Serial microsamples of blood were collected from an indwelling cannula in unanesthetized, freely moving rats at intervals of 2-60 min for up to 12 hr. The samples' CCl4 content was measured by headspace gas chromatography. Thereby, it was possible to obtain blood CCl4 concentration-versus-time profiles. Animals were euthanized 24 hr postdosing and blood was collected for measurement of serum enzymes as indices of hepatotoxicity. No toxicologically significant differences in pharmacokinetic parameters as a function of Emulphor concentration were found. Similarly the hepatotoxic potency of 10 and 180 mg/kg CCl4, as reflected by elevation in serum enzyme activities, did not vary significantly with the concentration of Emulphor utilized. Hence, it can be concluded that Emulphor, in concentrations as high as 10% (equivalent to 260 mg Emulphor/kg bw) in aqueous emulsions, does not significantly affect the absorption, disposition, or acute hepatotoxicity of CCl4 in male Sprague-Dawley rats.

Preparation and Evaluation of Sustained-Release Solid Dispersions of Drugs with Eudragit Polymers

Coevaporates of paracetamol and rifampicin with Eudragit polymers of different natures (anionic, cationic, and zwitterionic) were prepared. Determination of dissolution rate of these coevaporates in dissolution media simulating those of the gastrointestinal tract (GIT) revealed that the release rate of paracetamol is retarded from all the coevaporates studied. In this respect, Eudragit L100-SS shows the highest sustainment of drug release, while Eudragit E100 shows the lowest. Conversely, the release of rifampicin from its coevaporates with the anionic Eudragit S100 polymer is more retarded than the corresponding coevaporate with the zwitterionic Eudragit RL100 or from coevaporates with equal mixtures of the two polymers.Increasing the polymer weight fraction in rifampicin coevaporates with Eudragit S100 up to 0.5 resulted in a corresponding decrease in the dissolution rate. However, beyond this weight fraction, the polymer effect on the dissolution rate of the drug becomes minimized. The results confirmed that the process of dissolution of the two drugs from their coevaporates is a diffusion-controlled release process.The biological performance of paracetamol coevaporates was monitored in rabbits; paracetamol level in plasma was found to follow first-order kinetics. for all the investigated paracetamol coevaporates, the peak plasma level was less than 50 μg/ml compared to a value of 60, μg/ml for the drug per se. The coevaporates of the drug with Eudragit L100-55 showed slowest rates of absorption and elimination as well as greatest half-peak and half-life times. Biological peformance of rifampicin coevaporates was assessed in human subjects receiving a single oral dose equivalent to 300 mg of the drug. The results depicted sustainment of drug release as a function of polymer weight fraction. A strict correlation was shown to exist between the total amount of drug excreted during 24 hr post dosing of the coevaporates and its in vitro dissolution rate.The results depicted that paracetamol can be formulated in the form of a coevaporate with Eudragit L100-55 to prepare a more safe sustained-release formulation with minimal side effects, and also revealed the advantages of administration of rifampicin in the form of a coevaporate with Eudragit S100 (4:1) at a single oral dose equivalent to 600 mg of drug.

Effect of acetaminophen administration on hepatic glutathione compartmentation and mitochondrial energy metabolism in the rat

Changes in cell energy metabolism and mitochondrial dysfunction have been observed after acetaminophen administration. Because consumption of hepatic glutathione is closely related to acetaminophen toxicity, we investigated the kinetics of: 1. glutathione depletion in liver mitochondria and cytosol; 2. State 3 and 4 respiratory rates of succinate-supplemented mitochondria; 3. rate of ATP synthesis; 4. oligomycin-sensitive ATP hydrolase activity and passive proton conductivity of inside-out vesicles of the inner mitochondrial membrane; and 5. changes in hepatic and mitochondrial malondialdehyde in the rat after in vivo acetaminophen administration. Two hours after acetaminophen injection, hepatic glutathione decreased and malondialdehyde increased. In the same interval, an increase in both State 3 and 4 respiratory rates of succinate-supplemented mitochondria was observed. This was accompanied by a decrease in the rate of ATP synthesis and the P/O ratio and by an increase in the passive proton permeability of the inner mitochondrial membrane, which was insensitive to oligomycin. No significant change in oligomycin-sensitive ATP hydrolase activity was observed. Four hours after APAP injection, the respiratory rates, as well as the proton conductivity, decreased, the rate of ATP synthesis was restored, and the mitochondrial glutathione started to increase; the cytosolic levels of glutathione were still low and the cytosolic and mitochondrial levels of malondialdehyde remained high for 2 more hr. The concentrations of these indices were completely restored 24 hr postdosing. Our findings suggest that acetaminophen administration selectively depletes (within 2 hr) mitochondrial glutathione, and produces local toxicity by altering membrane permeability and decreasing the efficiency of oxidative phosphorylation. This renders mitochondria more susceptible to oxidative damage, especially during increased free radical production, as in the case of enhanced mitochondrial respiration in State 4. The concomitant restoration of mitochondrial respiration, oxidative phosphorylation, membrane permeability, and glutathione levels is consistent with the importance of the mitochondrial glutathione pool for the protection of the mitochondrial membrane against oxidative damage.

Urinary Excretion Kinetics of Pyrene and Benzo(a)pyrene Metabolites Following Intravenous Administration of the Parent Compounds or the Metabolites

The detailed urinary excretion profiles of 1-hydroxypyrene (1-OHP) and benzo(a)pyrene (BaP) metabolites were studied following acute intravenous administration of pyrene and BaP, respectively, or after injection of the metabolites themselves. Male Sprague–Dawley rats were exposed to 4 μmol 1-OHP/kg or 15 μmol pyrene/kg. Other rats were exposed to 2 μmol/kg of a mixture of four BaP metabolites (3-hydroxyBaP (3-OHBaP), 9-hydroxyBaP (9-OHBaP), trans-4,5-dihydrodiolBaP (4,5-diolBaP), and trans-9,10-dihydrodiol (9,10-diolBaP)) or 40 μmol BaP/kg. Urine samples were collected at frequent intervals over 48 or 96 hr. Injection of both pyrene and 1-OHP produced similar biphasic excretion profiles. An apparent first order half life of 6.9 and 6.6 hr, respectively, could be calculated for the second phase of elimination. Comparable 3-OHBaP excretion profiles were obtained after injection of BaP or a mixture of BaP metabolites. Elimination kinetics showed at least two steps, the second step having a first order apparent half life of 8.1 and 7.6 hr following BaP and BaP metabolites injection, respectively. Time profiles of 4,5-diolBaP excretion following administration of BaP or a mixture of BaP metabolites were almost identical. Elimination was linear and a first order apparent half life of 3.1 and 3.6 hr could be calculated. Elimination of 4,5-diolBaP was much more rapid than that of 3-OHBaP and complete within 24 hr postdosing. Therefore, results suggest that (1) phase I biotransformation is not the rate-limiting step in the excretion of 1-OHP, and 3-OHBaP and 4,5-diolBaP following injection of pyrene and BaP, respectively, and (2) similarities in the first order apparent half life of 3-OHBaP and 1-OHP for the late phase of excretion suggest that 1-OHP could be a good surrogate for 3-OHBaP.

Mesenteric arteriopathy in the rat induced by phosphodiesterase III inhibitors: an investigation of morphological, ultrastructural, and hemodynamic changes.

A reproducible model of a phosphodiesterase III (PDE III) inhibitor-induced arteriopathy has been developed in the rat after subcutaneous administration of SK&F 95654. Administration of this potent PDE III inhibitor induced an arteriopathy of mesenteric arteries within 24 hr that was dose-related in intensity and incidence over the range 0.174, 0.348, 0.523, and 0.697 mmol/kg. The arteriopathy was restricted to muscular arteries of external diameter of 100-800 microns and was shown microscopically to be focal or segmental medial necrosis and hemorrhage. A time-course experiment, conducted from 3 to 24 hr postdosing, showed that the first changes observed 6 hr postdosing were on the endothelium followed by focal hemorrhages into the media at 12 hr postdosing, causing compression, degeneration, and necrosis of myocytes. From 16 hr postdosing, there was focal endothelial cell necrosis and loss of confluence. Leukocytes and activated platelets were found adhering to exposed basement lamina and seen to pass through endothelial gaps into the subintima. By 24 hr postdosing, medial necrosis was extensive with large areas of media replaced by erythrocytes, cell debris, and a few leukocytes and platelets. The effect of 3 structurally dissimilar PDE III inhibitors administered subcutaneously at a dose of 0.697 mmol/kg was compared with that of SK&F 95654. The arteriopathy induced by these compounds were identical to that produced by SK&F 95654 with the incidence and severity of lesions ranked in the following order: SK&F 95654 > WIN 62582 > SK&F 94836, with no macroscopic lesions observed for SK&F 94120. Systolic blood pressure was measured for these 4 PDE III inhibitors at regular intervals over the 24-hr period postadministration by a plesthymographic method. The severity of the arterial lesions correlated with the magnitude of hypotension induced by these agents. It is postulated that the arterial damage is a consequence of profound vasodilation resulting in abnormal endothelial permeability and increased wall tension, resulting in progressive medial necrosis and hemorrhage.