Plasma Clearance Values Research Articles

Abstract 2607: Endoxifen pharmacokinetics and bioavailability of in female rats and dogs

Abstract Endoxifen (END), the active metabolite of tamoxifen (TAM), is currently being developed as a drug for the treatment of estrogen receptor positive breast cancer based on recent in vitro and clinical data demonstrating that TAM drug effect is substantially related to circulating plasma concentrations of END, a secondary metabolite produced by human CYP2D6 metabolism. Women with deficient CYP2D6 metabolism have lower END concentrations and a higher risk of breast cancer recurrence. We characterized the pharmacokinetics and oral bioavailability of END in female rats and dogs. We further compared END exposure following equivalent doses of END and TAM. Rats were administered 2 mg/kg i.v. END, 20 − 200 mg/kg oral END or 80 mg/kg oral TAM. Fasted dogs were administered 0.5 mg/kg i.v. END, 15 − 100 mg/kg oral END or 30 mg/kg oral TAM. Plasma END concentrations were also measured in rats or dogs after oral dosing once per day for 4 consecutive days. Plasma samples were analyzed using a validated reverse-phase HPLC method with fluorescence detection. In the single-dose administration study, terminal elimination half-life and plasma clearance values in rats given 2 mg/kg i.v. END were 6.3 h and 2.4 L/h/kg, respectively. Plasma concentrations above 0.1 µM and 1 µM END were achieved with 20 mg/kg and 200 mg/kg doses, respectively, and those concentrations were maintained for longer than 24 h. Oral absorption of END was linear over the range of 20 − 140 mg/kg with a bioavailability greater than 67%. END exposure in rats following 80 mg/kg oral END was 100-fold greater than END exposure following 80-mg/kg oral TAM. Following 0.5 mg/kg i.v. END to dogs, terminal elimination half-life and plasma clearance values were 9.2 h and 0.4 L/h/kg, respectively. Plasma concentrations above 1 µM and 10 µM were achieved with the 15 mg/kg dose and 100 mg/kg, respectively, and those concentrations were maintained for longer than 24 h. Absorption was linear over the range of 15 − 100 mg/kg with a bioavailability greater than 50%. END exposure in dogs following 30 mg/kg oral END was 10-fold greater than END exposure following 30-mg/kg oral TAM. In the repeated-dose administration study, the day 4 END peak plasma concentration in rats increased from 1 to 9 µM as the daily END dose was increased from 20 to 200 mg/kg. The day 4 END peak plasma concentration in dogs exceeded 20 µM and plasma concentrations remained above 2 µM for 24 hours following 4 daily doses of 30 mg/kg END. Based on these data, we conclude that END has high oral bioavailability; high concentrations may be maintained after repeated dosing in dogs and rats; and substantially higher END concentrations are achievable following an oral dose of END in comparison to a similar dose of TAM. Supported by NCI contract N01-CM-52206. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2607.

Abstract 3603: Endoxifen pharmacokinetics and bioavailability in female mice

Abstract In collaboration with NCI, we are developing endoxifen (END), the active metabolite of tamoxifen (TAM), as a drug for the treatment of estrogen receptor positive breast cancer. Tamoxifen treated women with reduced or absent CYP2D6 enzyme activity have significantly lower END plasma concentrations. In a retrospective analysis of two different prospective tamoxifen trials (NCCTG and ABCSG), we have demonstrated that genetic or drug-induced reductions in CYP2D6 metabolism are associated with a higher risk of breast cancer recurrence in Tam treated ER positive breast cancer. To test the hypothesis that clinically relevant concentrations and exposures of END are achieved following oral administration of END, we characterized the pharmacokinetics of i.v. and oral END and oral TAM in female mice. Endoxifen was synthesized and provided by NCI/DTP. Plasma samples were analyzed using a validated reverse-phase HPLC method with fluorescence detection. Following i.v. administration of a 1 mg/kg dose, peak plasma concentration, terminal elimination half-life and plasma clearance values were 0.26 µM, 6.5 h and 11.8 L/h/kg, respectively. Oral pharmacokinetics and bioavailability were determined following doses of 50 and 200 mg/kg. Peak plasma concentrations of 0.76 µM were achieved with a 50 mg/kg dose and plasma concentrations above 0.1 µM were maintained for longer than 8 h. Peak plasma concentrations of 8 µM were achieved with a 200 mg/kg dose and plasma concentrations above 2 µM were maintained for 24 h. Bioavailability was 50% and greater than 100% following the 50 mg/kg and 200 mg/kg doses, respectively. Based on pharmacokinetic data for oral END and TAM normalized to a 50 mg/kg oral dose, END plasma concentrations and total exposure were 20-fold greater following oral END as compared to oral TAM. In conclusion, END has high oral bioavailability in mice and substantial plasma concentrations are achieved and maintained after a single oral END dose. Compared to an equivalent oral dose of Tam, substantially higher END concentrations are achieved following oral END. These data suggest that the primary administration of End may overcome the limitations related to human CYP2D6 metabolism, and support the ongoing development of endoxifen as a primary therapy for ER breast cancer. Supported by the Mayo Comprehensive Cancer Center Grant (CA15083) and the Mayo Clinic Breast Cancer SPORE (CA 116201; MMA, JMR, MPG). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3603.

Abdominal irradiation modulates 5-Fluorouracil pharmacokinetics

BackgroundConcurrent chemoradiation with 5-fluorouracil (5-FU) is widely accepted for treatment of abdominal malignancy. Nonetheless, the interactions between radiation and 5-FU remain unclear. We evaluated the influence of abdominal irradiation on the pharmacokinetics of 5-FU in rats.MethodsThe radiation dose distributions of cholangiocarcinoma patients were determined for the low dose areas, which are generously deposited around the intrahepatic target volume. Then, corresponding single-fraction radiation was delivered to the whole abdomen of Sprague-Dawley rats from a linear accelerator after computerized tomography-based planning. 5-FU at 100 mg/kg was intravenously infused 24 hours after radiation. A high-performance liquid chromatography system equipped with a UV detector was used to measure 5-FU in the blood. Ultrafiltration was used to measure protein-unbound 5-FU.ResultsRadiation at 2 Gy, simulating the daily human treatment dose, reduced the area under the plasma concentration vs. time curve (AUC) of 5-FU by 31.7% compared to non-irradiated controls. This was accompanied by a reduction in mean residence time and incremental total plasma clearance values, and volume of distribution at steady state. Intriguingly, low dose radiation at 0.5 Gy, representing a dose deposited in the generous, off-target area in clinical practice, resulted in a similar pharmacokinetic profile, with a 21.4% reduction in the AUC. This effect was independent of protein binding capacity.ConclusionsAbdominal irradiation appears to significantly modulate the systemic pharmacokinetics of 5-FU at both the dose level for target treatment and off-target areas. This unexpected and unwanted influence is worthy of further investigation and might need to be considered in clinical practice.

In Vitro Hepatic Metabolism Explains Higher Clearance of Voriconazole in Children versus Adults: Role of CYP2C19 and Flavin-Containing Monooxygenase 3

Voriconazole is a broad spectrum antifungal agent for treating life-threatening fungal infections. Its clearance is approximately 3-fold higher in children compared with adults. Voriconazole is cleared predominantly via hepatic metabolism in adults, mainly by CYP3A4, CYP2C19, and flavin-containing monooxygenase 3 (FMO3). In vitro metabolism of voriconazole by liver microsomes prepared from pediatric and adult tissues (n = 6/group) mirrored the in vivo clearance differences in children versus adults, and it showed that the oxidative metabolism was significantly faster in children compared with adults as indicated by the in vitro half-life (T(1/2)) of 33.8 + or - 15.3 versus 72.6 + or - 23.7 min, respectively. The K(m) for voriconazole metabolism to N-oxide, the major metabolite formed in humans, by liver microsomes from children and adults was similar (11 + or - 5.2 versus 9.3 + or - 3.6 microM, respectively). In contrast, apparent V(max) was approximately 3-fold higher in children compared with adults (120.5 + or - 99.9 versus 40 + or - 13.9 pmol/min/mg). The calculated in vivo clearance from in vitro data was found to be approximately 80% of the observed plasma clearance values in both populations. Metabolism studies in which CYP3A4, CYP2C19, or FMO was selectively inhibited provided evidence that contribution of CYP2C19 and FMO toward voriconazole N-oxidation was much greater in children than in adults, whereas CYP3A4 played a larger role in adults. Although expression of CYP2C19 and FMO3 is not significantly different in children versus adults, these enzymes seem to contribute to higher metabolic clearance of voriconazole in children versus adults.

The UGT2B15*2 polymorphism is a major determinant of oxazepam clearance in human volunteers.

Oxazepam is specifically glucuronidated by UGT2B15. In previous studies we have shown that a common non‐synonymous polymorphism in the UGT2B15 gene (*2, D85Y) results in lower oxazepam glucuronidation by recombinant enzyme and human liver microsomes. The goal of this study was to determine the effect of UGT2B15*2 on the pharmacokinetics of oxazepam in 30 healthy male subjects administered 15 mg oxazepam by mouth. UGT2B15 genotype frequencies were 0.20, 0.67 and 0.13 for *1/*1, *1/*2 and *2/*2, respectively. Mean (+/‐ SD) plasma oxazepam area under the curve values extrapolated to infinity were significantly higher (P<0.001, ANOVA) in *2/*2 subjects (2.50 +/‐ 0.96 ug/mL*hr) compared with *1/*2 (1.37 +/‐ 0.40) and *1/*1 (0.981 +/‐ 0.29) subjects. Furthermore, mean weight‐normalized plasma clearance values were significantly lower (P=0.003, ANOVA) in *2/*2 subjects (1.55 +/‐ 0.61 mL/min/kg) compared with *1/*2 (2.56 +/‐ 0.74) and *1/*1 (3.53 +/‐ 1.14) subjects. These results mirror our previous in vitro results and substantiate the UGT2B15*2 polymorphism as a major determinant of oxazepam clearance in vivo. (Supported by NIH grants GM‐61834, DK‐79300, AI‐058784, and AG‐017880.)

Stereoselective pharmacokinetics of diniconazole enantiomers in rabbits

Diniconazole [(E)-(RS)-1-(2,4,-dichlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazole-1-yl)pent-1-en-3-ol)] is a potent triazole fungicide. The enantioselective pharmacokinetics of diniconazole enantiomers in rabbits was studied via intravenous (i.v.) injection. The pharmacokinetics and the enantiomer fraction (EF) were determined using normal high-performance liquid chromatography with diode array detection and a cellulose-tris-(3,5-dimethylphenylcarbamate)-based chiral stationary phase (CDMPC-CSP). The time-concentration curves in plasma were fitted by a two-compartment open mode. The results showed that the concentration of S-diniconazole in plasma decreased faster than that of R-diniconazole, and EFs increased with time after administration of racemic diniconazole (rac-diniconazole). The R-/S-enantiomer ratio of the area under the time-plasma concentration curve (AUC(0-infinity)) after administration was 1.52. The total plasma clearance value of S-enantiomer was 1.57-fold higher than that of the R-diniconazole. These results indicate substantial stereoselectivity in the kinetics of diniconazole enantiomers in rabbit.

In Vivo Pharmacokinetics of Hesperidin Are Affected by Treatment with Glucosidase-like BglA Protein Isolated from Yeasts

Hesperidin is an abundant flavanone glycoside in citrus fruits and has been reported to possess a wide range of biological activities. However, hesperidin has poor bioavailability. Here, we tested the hypothesis that hesperetin found in chenpi will have a better bioavailability than hesperidin and that treatment of hesperidin with the glucosidase-like yeast Bg1A protein will increase its bioavailability. The results indicate that hesperidin in pure or extract form is hydrolyzed by BglA protein extracted from Sporobolomyces singularis or expressed in Escherichia coli BL21 (DE3). This biotransformation affected the plasma pharmacokinetics of total hesperetin in rats, in that the plasma T max was significantly shorter after administration of BglA protein-treated hesperidin than after administration of hesperidin extract. In addition, the area under the curve values for total hesperetin after administration of Bg1A-treated hesperidin were approximately 4-fold higher by oral administration and 3-fold higher by intravenous administration, respectively. In contrast, the plasma clearance value and volume of distribution after administration of Bg1A-treated hesperidin extract or pure hesperetin were significantly smaller than after administration of untreated hesperidin extract or pure hesperidin. This is the first study that systemically determines the absolute bioavailability of hesperidin and hesperetin simultaneously, shows clearly that hesperetin is more bioavailable than hesperidin regardless of the route of administration, and shows that prior transformation of hesperidin to hesperetin via fermentation should significantly increase its bioavailability because of the action of the yeast glycosidase-like protein BglA.

Pharmacokinetics of intravenous dexmedetomidine in children under 11 yr of age

BackgroundInformation has been very limited on the pharmacokinetics of the selective α2-adrenoceptor agonist dexmedetomidine in children, particularly in children <2 yr of age. MethodsEight children aged between 28 days and 23 months and eight children aged between 2 and 11 yr undergoing either elective bronchoscopy or nuclear magnetic resonance imaging were included in the study. Dexmedetomidine 1 μg kg−1 was infused i.v. over 5 min. Blood samples for the measurement of plasma concentrations of dexmedetomidine were collected for 5 h after starting the infusion. Pharmacokinetic calculations were based on non-compartmental methods. ResultsIn the two groups of paediatric patients, the median (range) values for total plasma clearance of dexmedetomidine were 17.4 (14.1–27.6) and 17.3 (9.3–22.5) ml kg−1 min−1, for volume of distribution at steady state 3.8 (1.9–4.6) and 2.2 (1.3–2.8) litre kg−1 (P<0.05), and for elimination half-life 139 (90–198) and 96 (69–140) min (P<0.05), respectively. The volume of distribution at steady state was negatively associated with subject age (r=−0.69, P<0.05). ConclusionsTo reach a certain plasma concentration, children younger than 2 yr of age evidently need larger initial doses of dexmedetomidine than the older children, as young children have a larger volume of distribution of the drug than older children and adults. Since the total plasma clearance of dexmedetomidine is independent of age, similar rates of infusion can be used in younger and older children to maintain a steady-state concentration of dexmedetomidine in plasma.

Effect of the anticoagulant ethylenediamine tetra-acetic acid (EDTA) on the estimation of pharmacokinetic parameters: A case study with tigecycline and ciprofloxacin

Tigecycline and ciprofloxacin were employed as the model compounds to study the effect of the anticoagulant ethylenediamine tetra-acetic acid (EDTA), which is used during plasma sample preparations, on the determination of pharmacokinetic parameters. The pharmacokinetic parameters were determined in rats following intravenous infusion with blood samples collected in serum separators, with either EDTA- or heparin-coated tubes. The blood-to-plasma (B:P) partition ratio and plasma protein binding were determined in vitro in rat or human blood collected in either EDTA- or heparin-coated tubes. Drug concentrations were quantified by liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) analysis. In tigecycline-treated rats drug concentrations were twofold lower in EDTA plasma, leading to a twofold lower area under plasma concentration–time curve (AUC) and twofold higher plasma clearance values as compared with those obtained from heparin plasma. No differences were noted in the pharmacokinetic parameters obtained from heparin-treated plasma versus serum. The B:P partition ratio and unbound fraction for tigecycline were significantly higher in EDTA-treated blood. When normalized to the B:P partition ratios, the tigecycline blood clearance values were identical between samples collected in EDTA- or heparin-coated tubes. Similar but smaller differences were observed for ciprofloxacin. It was concluded that EDTA might compete with tigecycline and ciprofloxacin for chelating metal ions and thus affect drug partition between blood and plasma compartments, leading to inaccurate measurement of pharmacokinetic parameters in plasma.

A phase 1, placebo-controlled, randomised, double-blind (within dose panels) study evaluating the safety, tolerability and pharmacokinetics of intravenous NXY-059 in Japanese subjects

ABSTRACTObjective: NXY‑059 has a proposed mechanism of action of free-radical trapping and has been studied in clinical trials based on positive effects seen in experimental models of acute ischaemic stroke. This study evaluated the safety, tolerability and pharmacokinetics of NXY‑059 in healthy Japanese male subjects compared with previous data from healthy Caucasian subjects.Research design and methods: The primary objective of this phase 1, double-blind, randomised, placebo-controlled, dose-escalating study was to evaluate the safety and tolerability of an 8- or 24‑h intravenous infusion of NXY-059 targeting an unbound plasma concentration of 25–300 μmol/L in healthy Japanese male subjects (20–45 years). NXY‑059 pharmacokinetics were also assessed, and any differences in pharmacokinetics between Japanese and previously studied Caucasian subjects (20–45 years) were explored.Results: NXY‑059 was generally well tolerated and no safety concerns were identified. There was a similar incidence of adverse events between subjects receiving NXY‑059 or placebo, and no obvious trend towards an increased incidence of adverse events with increasing doses of NXY‑059 was observed. In addition, there was no evidence of any vasoirritative effects or changes in renal function. The tolerability profile was similar in Caucasian subjects. The pharmacokinetic results indicate proportional exposure of 8‑h and 24‑h infusions of NXY‑059 resulting in mean unbound steady state plasma concentrations up to 417 μmol/L and 379 μmol/L, respectively. Plasma clearance values for NXY‑059 were similar in both Japanese and Caucasian subjects.Conclusions: This study suggests that the tolerability and pharmacokinetics of NXY‑059 in healthy Japanese male subjects and Caucasians are similar.

Preclinical pharmacokinetics and in vitro metabolism of dasatinib (BMS-354825): a potent oral multi-targeted kinase inhibitor against SRC and BCR-ABL

Dasatinib (BMS-354825), a potent oral multi-targeted kinase inhibitor against SRC and BCR-ABL, has recently been approved for the treatment of chronic myelogenous leukaemia (CML) in imatinib-acquired resistance and intolerance. In vitro and in vivo studies were conducted to characterize the pharmacokinetics and metabolism of dasatinib in mouse, rat, dog, and monkey. Possible mechanisms contributing to the incomplete oral bioavailability of dasatinib in animals were investigated. Metabolic stability of dasatinib was measured after incubation with liver microsomes (either NADPH- or UDPGA-fortified) and isolated hepatocytes obtained from mouse, rat, dog, monkey, and human. In all cases, substrate depletion over time was measured, and appropriate scaling factors were used to predict in vivo clearance. Pharmacokinetics of dasatinib were determined in mice, rats, dogs, and monkeys after administration of single intravenous or oral doses. In addition, the routes of excretion were investigated after administration of dasatinib to bile duct cannulated (BDC) rats. Absorption and first-pass metabolism were evaluated as possible reasons for the incomplete oral bioavailability using various in vitro and in vivo models like Caco-2 cells, P-glycoprotein (P-gp) knockout mice, and intra-portal dosing in rats. In vivo systemic plasma clearance values of dasatinib were 62, 26, 25, and 34 ml/min/kg in mouse, rat, dog, and monkey, respectively. Scaling of in vitro hepatocyte and liver microsomal data gave reasonably good predictions of in vivo clearances across all species. Percent distribution in blood cells ranged from 43% in mouse to 57% in dog. Dasatinib showed high volumes of distribution (>3 l/kg) and high serum protein binding values (>90%) in all four species tested. Oral bioavailability of dasatinib ranged from 14% in the mouse to 34% in the dog. In rats, bioavailability after an intraportal dose was comparable to that after intra-arterial administration. In BDC rats, less than 15% of an intravenous dose was excreted unchanged in urine, bile, and the gastrointestinal tract, suggesting that dasatinib is cleared primarily via metabolism. Dasatinib has high intrinsic permeability in Caco-2 cells, however, the efflux ratio was approximately two-fold indicating that it may be a substrate for an intestinal efflux transporter. However, in vivo studies in P-gp knockout mice versus wild-type mice showed no difference in the amount of dasatinib remaining unabsorbed in the gastrointestinal tract, suggesting that P-gp may not be responsible for the incomplete bioavailability. Dasatinib shows intermediate clearance in mouse, rat, dog, and monkey, and distributes extensively in those species. Oxidative metabolism appears to be the predominant clearance pathway. The incomplete oral bioavailability may be due to both incomplete absorption and high first-pass metabolism. However, the efflux transporter, P-glycoprotein does not appear to be limiting oral absorption.

Pharmacokinetics of florfenicol, thiamphenicol, and chloramphenicol in turkeys

The pharmacokinetics of florfenicol (FF), thiamphenicol (TP) and chloramphenicol (CP) after single intravenous (i.v.) or oral (p.o.) administration was studied in an independent cross-over study in broiler turkeys. All the fenicol antibiotics were administered at a dose of 30 mg/kg b.w. and their concentrations in plasma samples were assayed using the same validated high-performance liquid chromatography method. Pharmacokinetic parameters were calculated by a noncompartmental method. The kinetic profiles of the compounds were compared with the results of the structure-activity relationship. According to the proposed mathematical description, no differences in plasma clearance values for the studied antibiotics were observed. The mean residence time values of FF, TF, and CP after i.v. injection were 3.37+/-0.63, 2.43+/-0.29, and 2.12+/-0.21 h, respectively. The mean values of Varea for FF (1.39+/-0.31 L/kg) and TP (1.31+/-0.19 L/kg) were similar, but significantly different from that of CP (1.04+/-0.12 L/kg). The bioavailabilities of FF, TP, and CP after oral administration were 82%, 69%, and 45%, respectively. Differences in the bioavailability values of the compared fenicol antibiotics correspond to the ratio of the apolar/polar surface areas of their particles.

Metabolism, Disposition and Pharmacokinetics of PR-171, a Novel Inhibitor of the 20S Proteasome.

Clinical studies using the boronic acid-based proteasome inhibitor bortezomib have validated the proteasome as a therapeutic intervention point for the treatment of multiple myeloma and non-Hodgkin's Lymphoma. PR-171 is a novel peptide epoxyketone inhibitor of the 20S proteasome that is currently in phase I clinical trials. Herein, the metabolism, disposition and pharmacokinetics of PR-171 are described. Incubation of 3H-PR-171, labeled in the phenylalanine ring, with freshly prepared rat, monkey and human hepatocyte preparations showed similar metabolite profiles with six predominant radiolabeled metabolites observed in all three species; no metabolites unique to human hepatocytes were detected. The diol of PR-171, formed via epoxide ring hydrolysis, was the most abundant observed metabolite in hepatocyte preparations of each of the three species. Single dose IV bolus PK studies using unlabeled PR-171 in Sprague Dawley rats (2 to 9 mg/kg) and cynomolgus monkeys (1 to 4 mg/kg) showed approximately dose linear PR-171 exposure for both species as measured by plasma AUCs. In both species, PR-171 plasma clearance values (554 and 227 mL/min/kg in rat and monkey, respectively) exceeded hepatic blood flow implying that extrahepatic clearance mechanism(s) must be involved. While the in vitro hepatocyte results suggest that hepatic clearance mechanism(s) are also likely to be involved, in vivo hepatic clearance cannot be conclusively inferred from available data. As predicted by the in vitro hepatocyte results, PR-171 diol was detected at abundant levels in plasma from both rat and monkey. Human pharmacokinetics are similar to those of rat and monkey. Mass balance and tissue distribution were also investigated in rats using 3H-PR-171. Radioactivity was widely distributed following a single IV bolus dosing and reached maximum concentrations at times ranging from 0.5 to 24 hours in different tissues. At 168 hours post-dose, more than 44% of the administrated radioactivity remained in tissues. Excretion via urine and feces accounted for 14% and 18%, respectively, of administered radiation. The distribution of radioactivity may not be indicative of the true distribution of PR-171 as much of the radioactivity may reflect metabolic incorporation of the radiolabel and/or 3H exchange with water.

Extrapolating from animal studies to the efficacy in humans of a pretreatment combination against organophosphate poisoning

The extrapolation from animal data to therapeutic effects in humans, a basic pharmacological issue, is especially critical in studies aimed to estimate the protective efficacy of drugs against nerve agent poisoning. Such efficacy can only be predicted by extrapolation of data from animal studies to humans. In pretreatment therapy against nerve agents, careful dose determination is even more crucial than in antidotal therapy, since excessive doses may lead to adverse effects or performance decrements. The common method of comparing dose per body weight, still used in some studies, may lead to erroneous extrapolation. A different approach is based on the comparison of plasma concentrations at steady state required to obtain a given pharmacodynamic endpoint. In the present study, this approach was applied to predict the prophylactic efficacy of the anticholinergic drug caramiphen in combination with pyridostigmine in man based on animal data. In two species of large animals, dogs and monkeys, similar plasma concentrations of caramiphen (in the range of 60-100 ng/ml) conferred adequate protection against exposure to a lethal-dose of sarin (1.6-1.8 LD(50)). Pharmacokinetic studies at steady state were required to achieve the correlation between caramiphen plasma concentrations and therapeutic effects. Evaluation of total plasma clearance values was instrumental in establishing desirable plasma concentrations and minimizing the number of animals used in the study. Previous data in the literature for plasma levels of caramiphen that do not lead to overt side effects in humans (70-100 ng/ml) enabled extrapolation to expected human protection. The method can be applied to other drugs and other clinical situations, in which human studies are impossible due to ethical considerations. When similar dose response curves are obtained in at least two animal models, the extrapolation to expected therapeutic effects in humans might be considered more reliable.

LC–MS/MS assay and dog pharmacokinetics of the dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501)

The dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501) has potent in vitro cytotoxicity and in vivo antitumor activity. SJG-136 binds in the minor groove of DNA and produces G-G interstrand cross-links via reactive N(10)-C(11)/N(10')-C(ll') imine/carbinolamine moieties. We have developed a sensitive, specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method for the quantitative determination of SJG-136 in plasma. SJG-136 was isolated by solid phase extraction through a C8 column, reverse-phase HPLC separation was accomplished on a C18 column with isocratic elution and MS/MS detection, monitoring the m/z 557-m/z 476 transition after electrospray ionization. The linear range and lower limit of quantitation from plasma standard curves were 2.8-1800 nM, and 5 nM, respectively. SJG-136 plasma protein binding was species-dependent. Values of the unbound fraction in human, rat and mouse were 25%, 16.2% and <1%, respectively. Protein binding was saturable in dog plasma where the unbound fraction increased from 10.8% to 22.3% over a 22-720 nM concentration range. SJG-136 pharmacokinetics after a single intravenous dose were best fit to a two-compartment open model with elimination half-life and plasma clearance values of 97 min and 6.1 mL/min/kg, respectively. SJG-136 did not accumulate in plasma following intravenous administration of 1.0 microg/kg doses for five consecutive days.

Enantioselective degradation kinetics of metalaxyl in rabbits

Metalaxyl [methyl- N-(2′-methoxyacetyl)- N-(2,6-dimethylphenyl)- d, l- alaninate] is a potent phenylamide fungicide. The (−)-( R)-isomer accounts for most of the fungicidal activity. A possible stereo and/or enantioselective kinetics of metalaxyl in rabbits was investigated by intravenous injection. The concentrations of (−)-( R)- and (+)-( S)-metalaxyl in plasma, liver, and kidney tissue were determined by HPLC with a cellulose–Tris-(3,5-dimethylphenylcarbamate)-based chiral stationary phase and gas chromatography-mass spectroscopy. After intravenous administration of racemic metalaxyl (40 mg/kg), the (+)-( S)-enantiomer levels in plasma, liver, and kidney decreased more rapidly than the (−)-( R)-isomer. The area ratio of the (−)-( R)-/(+)-( S)-enantiomer under the concentration–time curve (AUC 0 → ∞ ) in plasma after drug application was 1.62. The total plasma clearance value of the (+)-( S)-enantiomer was 1.53 and higher than that of the (−)-( R)-enantiomer. The [ R]/[ S] ratio in plasma was >1 for standard rac-metalaxyl at each time point. The other pharmacokinetic parameters of the enantiomers were also different. The results indicate substantial stereoselectivity in the degradation of metalaxyl enantiomers in rabbits.

Spiro and Dispiro-1,2,4-trioxolanes as Antimalarial Peroxides: Charting a Workable Structure−Activity Relationship Using Simple Prototypes

This paper describes the discovery of synthetic 1,2,4-trioxolane antimalarials and how we established a workable structure-activity relationship in the context of physicochemical, biopharmaceutical, and toxicological profiling. An achiral dispiro-1,2,4-trioxolane (3) in which the trioxolane is flanked by a spiroadamantane and spirocyclohexane was rapidly identified as a lead compound. Nonperoxidic 1,3-dioxolane isosteres of 3 were inactive as were trioxolanes without the spiroadamantane. The trioxolanes were substantially less effective in a standard oral suspension formulation compared to a solubilizing formulation and were more active when administered subcutaneously than orally, both of which suggest substantial biopharmaceutical liabilities. Nonetheless, despite their limited oral bioavailability, the more lipophilic trioxolanes generally had better oral activity than their more polar counterparts. In pharmacokinetic experiments, four trioxolanes had high plasma clearance values, suggesting a potential metabolic instability. The toxicological profiles of two trioxolanes were comparable to that of artesunate.

Physiological importance of SR-BI in the in vivo metabolism of human HDL and LDL in male and female mice

The physiological role of murine scavenger receptor class B type I (SR-BI) was evaluated by in vivo clearances of human HDL3 and LDL in normal and SR-BI knockout (KO) mice. In normal mice, cholesteryl esters (CEs) were removed faster than proteins, indicating a selective uptake process from both HDL3 and LDL. SR-BI KO mice showed 80% losses of HDL-CE selective uptake and the complete loss of LDL-CE selective uptake in the first phase of clearance. However, the second phase was characterized by an acceleration of CE disappearance in SR-BI KO mice. Thus, SR-BI is the only murine receptor mediating HDL-CE selective uptake, whereas a SR-BI-independent pathway specific to LDL can rescue SR-BI deficiency. The analysis of LDL recovered 3 h after injection in mice from different genotypes revealed that LDLs are significantly depleted in CE (reduction from 19% to 50% of the CE/protein ratios). A smaller LDL size in comparison with that of noninjected LDL was also detectable but was more evident for LDL recovered from normal mice. All LDL preparations migrate faster than noninjected LDL on agarose-barbital gels. Thus, both SR-BI-dependent and -independent pathways lead to substantial changes in LDL.

Pharmacokinetics of Guanfu Base I, an Active Metabolite of Guanfu Base A, in Rats

A liquid chromatographic-mass spectrometric (LC-MS) method was developed and validated for determination of guanfu base I (GFI), and the pharmacokinetics of GFI in Sprague-Dawley rats was examined. The method was linear in the 0.05-20 mug/ml concentration range (r=0.9994). The recovery of guanfu base I was more than 80%. The intraday and interday precision, expressed as the relative standard deviation (RSD), was generally good (<15%). After i.v. dosing, plasma GFI concentration declined in a bi-phasic manner with a terminal elimination half-life of 2.49 h. The total plasma clearance values was 1.46 l/h/kg. After oral dosing, the plasma GFI concentration reached a maximum within 0.5 h. The absolute bioavailability of GFI was 71.31%.

Stereoselective kinetic study of hexaconazole enantiomers in the rabbit

Hexaconazole [(RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol-1-yl)hexan-2-ol] is a potent triazole fungicide. The (-) isomer accounts for most of the fungicidal activity. The stereo- and/or enantioselective kinetics of hexaconazole were investigated in rabbits by intravenous injection. The concentrations of (-)- and (+)-hexaconazole in plasma, liver, and kidney tissue were determined by HPLC with a cellulose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phase and by gas chromatography-mass spectrometry. After intravenous administration of racemic hexaconazole (rac-hexaconazole) at 30 mg/kg, plasma, liver, and kidney levels of the (+)-enantiomer decreased more rapidly than those of the (-)-enantiomer. The (-)-/(+)-enantiomer ratio of the area under the concentration-time curve (AUC(0-infinity)) was 1.35. The total plasma clearance value (CL) of (+)-enantiomer was more than 1.3-fold higher than that of the (-)-hexaconazole. The enantiomeric ratio (ER) increased with time in plasma, liver, and kidney. Other pharmacokinetic parameters of the enantiomers were also different. These results indicate substantial stereoselectivity in the kinetics of hexaconazole enantiomers in rabbits.