A pharmacokinetic framework describing antibiotic adsorption to cardiopulmonary bypass devices.

Cardiopulmonary bypass (CPB) can alter pharmacokinetic (PK) parameters and the drug may adsorb to the CPB device, altering exposure. Cefazolin is a beta-lactam antibiotic used for antimicrobial prophylaxis during cardiac surgery supported by CPB. Adsorption of cefazolin could result in therapeutic failure. An ex vivo study was undertaken using CPB devices primed and then dosed with cefazolin and samples were obtained over 1 hour of recirculation. Twelve experimental runs were conducted using different CPB device sizes (neonate, infant, child, and adult), device coatings (Xcoating™, Rheoparin®, PH.I.S.I.O), and priming solutions. The time course of saturable binding, using Bmax (binding capacity), Kd (dissociation constant), and T2off (half-time of dissociation), described cefazolin adsorption. Bmax estimates for the device sizes were neonate 40.0 mg (95% CI 24.3, 67.4), infant 48.6 mg (95% CI 5.97, 80.2), child 77.8 mg (95% CI 54.9, 103), and adult 196 mg (95% CI 191, 199). The Xcoating™ Kd estimate was 139 mg/L (95% CI 27.0, 283) and the T2off estimate was 98.4 min (95% CI 66.8, 129). The Rheoparin® and PH.I.S.I.O coatings had similar binding parameters with Kd and T2off estimates of 0.169 mg/L (95% CI 0.01, 1.99) and 4.94 min (95% CI 0.17, 59.4). The Bmax was small (< 10%) relative to a typical total patient dose during cardiac surgery supported by CPB. A dose adjustment for cefazolin based solely on drug adsorption is not required. This framework could be extended to other PK studies involving CPB.

Subcutaneous nicotine delivery via needle-free jet injection: A porcine model.

Subcutaneous delivery of nicotine was performed using a novel electrically-operated needle-free jet injector, and compared to hypodermic needle delivery in a porcine model. Nicotine was delivered as a single, one-milligram dose into the abdominal skin, formulated as a 50 microliter aqueous solution. Plasma levels of nicotine and cotinine, its main metabolite, were then monitored over 2 h, following which the injection site was excised for histological examination. No irritation or tissue damage were found at the injection sites, and the jet-injected nicotine exhibited comparable absorption into the systemic circulation to that injected using a conventional needle and syringe. The needle-free jet injection of nicotine is a promising and well tolerated method. The data presented from this porcine model will support a first in human trial towards a new promising nicotine replacement therapy.

Varenicline improves motor and cognitive symptoms in early Huntington's disease.

The aim of this study was to describe the effects of varenicline, a smoking cessation aid that acts as a nicotinic agonist, on cognitive function in patients with early clinical Huntington's disease (HD) who were current smokers. Three gene-positive patients transitioning to symptomatic HD were evaluated using the Unified Huntington's Disease Rating Scale part I and III (motor and behavioral subscales) at baseline and after 4 weeks of treatment. Cognitive function was assessed using a touch screen computer-based neurocognitive test battery (IntegNeuro®). Varenicline (1 mg twice daily) significantly improved performance in executive function and emotional recognition tasks. Our case reports describe no clinically significant adverse effects and suggest that varenicline improves aspects of cognitive function in patients with early HD. A randomized controlled study is now underway.

Glucuronidation of anticancer prodrug PR-104A: species differences, identification of human UDP-glucuronosyltransferases, and implications for therapy.

PR-104, the phosphate ester of a dinitrobenzamide mustard [PR-104A; 2-((2-bromoethyl)-2-{[(2-hydroxyethyl) amino] carbonyl}-4,6-dinitroanilino)ethyl methanesulfonate], is currently in clinical trial as a hypoxia- and aldo-keto reductase 1C3 (AKR1C3)-activated prodrug for cancer therapy. Here, we investigate species (human, dog, rat, mouse) differences in metabolism to the corresponding O-glucuronide, PR-104G, and identify the human UDP-glucuronosyltransferase (UGT) isoforms responsible. After intravenous PR-104, plasma area under the concentration-time curve ratios (PR-104G/PR-104A) decreased in the order of dog (2.3) > human (1.3) > mouse (0.03) > rat (0.005). The kinetics of uridine 5'-diphosphoglucuronic acid-dependent glucuronidation by liver microsomes in vitro fitted the single-enzyme Michaelis-Menten equation with similar K(m) (∼150 µM) but differing V(max) (472, 88, 37, and 14 nmol/h/mg for dog, human, rat, and mouse, respectively), suggesting that facile glucuronidation is responsible for the anomalously rapid clearance of PR-104A in dogs. In vitro-in vivo extrapolation of PR-104A glucuronidation kinetics is consistent with this also being a major clearance pathway in humans. Recombinant UGT screening identified UGT2B7 as the only commercially available human isoform able to conjugate PR-104A, and UGT2B7 protein concentrations were highly correlated (r = 0.93) with PR-104A glucuronidation by liver microsomes from 24 individuals. The active hydroxylamine metabolite of PR-104A, PR-104H, was also glucuronidated by UGT2B7, although with slightly lower specificity and much lower rates. UGT2B7 mRNA expression was highly variable in human tumor databases. Glucuronidation of PR-104A greatly suppressed nitroreduction by AKR1C3 and NADPH-supplemented anoxic human liver S9 (9000g postmitochondrial supernatant). In conclusion, PR-104A is glucuronidated by UGT2B7 with high specificity and seems to make a major contribution to clearance of PR-104A in humans, but it also has the potential to confer resistance in some human tumors.

Comparative protein binding, stability and degradation of satraplatin, JM118 and cisplatin in human plasma in vitro.

1. Satraplatin is an investigational orally administered platinum-based antitumour drug. The present study compared the plasma protein binding, stability and degradation of satraplatin with that of its active metabolite JM118 and cisplatin. 2. The platinum complexes were incubated in human plasma for up to 2 h at 37 degrees C and quantified in plasma fractions by inductively coupled plasma-mass spectrometry on- or off-line to high-performance liquid chromatography. 3. All three platinum drugs became irreversibly bound to plasma proteins and showed negligible reversible protein binding. They were also unstable in plasma and generated one or more platinum-containing degradation products during their incubation. However, the three platinum complexes differed in the kinetics of their instability and protein binding, as well as in the number of degradation products formed during their incubation. 4. In conclusion, the plasma protein binding, instability and degradation of satraplatin and its active metabolite JM118 are qualitatively similar to that of cisplatin and other clinically approved platinum-based drugs. Quantitative differences in their irreversible protein binding and degradation were related to their respective physiochemical properties and bioactivation mechanisms.

Influence of mustard group structure on pathways of in vitro metabolism of anticancer N-(2-hydroxyethyl)-3,5-dinitrobenzamide 2-mustard prodrugs.

The dinitrobenzamide mustards are a class of bioreductive nitro-aromatic anticancer prodrugs, of which a phosphorylated analog (PR-104) is currently in clinical development. They are bioactivated by tumor reductases to form DNA cross-linking cytotoxins. However, their biotransformation in normal tissues has not been examined. Here we report the aerobic in vitro metabolism of three N-(2 hydroxyethyl)-3,5-dinitrobenzamide 2-mustards and the corresponding nonmustard analog in human, mouse, rat, and dog hepatic S9 preparations. These compounds have a range of mustard structures (-N(CH(2)CH(2)X)(2) where X = H, Cl, Br, or OSO(2)Me). Four metabolic routes were identified: reduction of either nitro group, N-dealkylation of the mustard, plus O-acetylation, and O-glucuronidation of the hydroxyethyl side chain. Reduction of the nitro group ortho to the mustard resulted in intramolecular alkylation and is considered to be an inactivation pathway, whereas reduction of the nitro group para to the mustard generated potential DNA cross-linking cytotoxins. N-Dealkylation inactivated the mustard moiety but may result in the formation of toxic acetaldehyde derivatives. Increasing the size of the nitrogen mustard leaving group abrogated the ortho-nitroreduction and N-dealkylation routes and thereby improved overall metabolic stability but had little effect on aerobic para-nitroreduction. All four compounds underwent O-glucuronidation of the hydroxyethyl side chain and further studies to elucidate the relative importance of this pathway in vivo are in progress.

Hepatic nitroreduction, toxicity and toxicokinetics of the anti-tumour prodrug CB 1954 in mouse and rat.

5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954), a promising anti-tumour compound, is associated with clinical hepatotoxicity. We have previously demonstrated that human liver preparations are capable of endogenous 2- and 4-nitroreduction of CB 1954 to generate highly potent cytotoxins. The present study initially examined the in vitro metabolism of CB 1954 in S9 preparations of several non-clinical species and strains. The CD-1 nu/nu mouse and Sprague-Dawley rat were subsequently chosen for further assessment of in vivo metabolism and hepatotoxicity of CB 1954, as well as the mechanisms that may be involved. Animals were administered the maximum tolerated dose (MTD). At 562 micromol/kg, the mouse exhibited transaminase elevation and centrilobular hepatocyte injury. Moreover, thiol adducts as well as hepatic glutathione depletion paralleled temporally by maximal nitroreduction were observed. The rat had a much lower MTD of 40 micromol/kg and showed signs of gastro-intestinal disturbances. In contrast to mouse, peri-portal damage and biliary changes were observed in rat without any alterations in plasma biomarkers or hepatic glutathione levels. Immunohistochemical analysis did not reveal any correlation between the location of injury and expression of cytochrome P450 reductase and NAD(P)H quinone oxidoreductase 1, two enzymes implicated in the bioactivation of this drug. In conclusion, the present study showed that following administration of CB 1954 at the respective MTDs, hepatotoxicity was observed in both mouse and rat. However, the degree of sensitivity to the drug and the mechanisms of toxicity involved appear to be widely different between CD-1 nu/nu mice and Sprague-Dawley rats.

Satraplatin activation by haemoglobin, cytochrome C and liver microsomes in vitro.

BACKGROUND: Satraplatin is thought to require reduction to a reactive Pt(II) complex (JM118) before exerting chemotherapeutic activity. In this study, we investigated the role of heme proteins in this reductive activation of satraplatin. METHODS: Satraplatin was incubated in solution with heme proteins and liver microsomes. The oxidation state of heme iron was monitored by visible absorption spectrometry. Satraplatin and JM118 were detected using a sensitive and specific HPLC-ICPMS assay. RESULTS: Satraplatin was stable in solutions containing haemoglobin, cytochrome c, glutathione, liver microsomes or NADH alone. However, in solutions containing haemoglobin plus NADH, satraplatin disappeared with a half-life of 35.8 mins. Under these conditions, satraplatin was reduced to JM118 and haemoglobin was oxidised to methaemoglobin. The reaction between haemoglobin and satraplatin was inhibited by carbon monoxide or by cooling the reaction solution. Cytochrome c and liver microsomes also reduced satraplatin to JM118 in a manner that depended upon the presence of NADH and was inhibited by carbon monoxide. CONCLUSION: This study has identified a mechanism of satraplatin activation involving metal-containing redox proteins and the transfer of electrons to the Pt(IV) drug from protein-complexed metal ions. Heme proteins may act by this mechanism as reducing agents for the activation of satraplatin in vivo.

Aerobic 2- and 4-nitroreduction of CB 1954 by human liver.

5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) is an anti-tumour prodrug which recently entered clinical trials in combination with Escherichia coli nitroreductase in a gene-directed enzyme prodrug therapy (GDEPT) context. A Phase I trial of the prodrug, however, revealed dose-limiting hepatotoxicity (transaminitis). The aim of this study was to find out whether the prodrug undergoes reductive metabolism in human liver to cytotoxic metabolites which may contribute to this clinical toxicity. CB 1954 (2.5-250 microM) was incubated with human liver preparations (2-8 mg/mL of S9, cytosolic or microsomal proteins) in the presence of NAD(P)H (1 mM). The NADH- and NADPH-dependent formation of both 2- and 4-nitroreduction products was demonstrated, with NADPH being the preferred cofactor, by HPLC and mass spectrometry. The major metabolite formed in all three human liver preparations was the 4-hydroxylamine, a potent DNA cross-linking cytotoxin. The 2-hydroxylamine and 2-amine metabolites were also detected, both of which have also been demonstrated to be highly cytotoxic. 2-Nitroreduction was far greater in S9 compared with cytosol and was not detected in microsomal preparations. Although 2- and 4-nitroreduction of CB 1954 was inhibited under hyperoxic conditions, substantial metabolism was observed under atmospheric oxygen levels. These studies demonstrate that human liver is capable of aerobic reductive bioactivation of CB 1954 to cytotoxic metabolites in vitro, possibly involving multiple enzymes, which may account for the clinical hepatotoxicity observed.

3'-azido-3'-deoxythymidine (AZT) induces apoptosis and alters metabolic enzyme activity in human placenta.

The anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) is the drug of choice for preventing maternal-fetal HIV transmission during pregnancy. Our aim was to assess the cytotoxic effects of AZT on human placenta in vitro. The mechanisms of AZT-induced effects were investigated using JEG-3 choriocarcinoma cells and primary explant cultures from term and first-trimester human placentas. Cytotoxicity measures included trypan blue exclusion, MTT, and reactive oxygen species (ROS) assays. Apoptosis was measured with an antibody specific to cleaved caspase-3 and by rescue of cells by the general caspase inhibitor Boc-D-FMK. The effect of AZT on the activities of glutathione-S-transferase, beta-glucuronidase, UDP-glucuronosyl transferase, cytochrome P450 (CYP) 1A, and CYP reductase (CYPR) in the placenta was assessed using biochemical assays and immunoblotting. AZT increased ROS levels, decreased cellular proliferation rates, was toxic to mitochondria, and initiated cell death by a caspase-dependent mechanism in the human placenta in vitro. In the absence of serum, the effects of AZT were amplified in all the models used. AZT also increased the amounts of activity of GST, beta-glucuronidase, and CYP1A, whereas UGT and CYPR were decreased. We conclude that AZT causes apoptosis in the placenta and alters metabolizing enzymes in human placental cells. These findings have implications for the safe administration of AZT in pregnancy with respect to the maintenance of integrity of the maternal-fetal barrier.

Metabolizing enzyme localization and activities in the first trimester human placenta: the effect of maternal and gestational age, smoking and alcohol consumption.

BACKGROUND: The rationale for this study was to assess the expression, activity and localization of the enzymes uridine diphosphate glucuronosyltransferase (UGT), beta-glucuronidase, cytochrome P450 1A (CYP1A) and cytochrome P450 2E1 (CYP2E1) in first trimester human placenta and to gauge the effects of maternal variables on placental metabolism. METHODS: CYP1A, CYP2E1, UGT and beta-glucuronidase activities were assessed in 25 placentas using ethoxyresorufin, chlorzoxazone, 4-methylumbelliferone and 4-methylumbelliferone glucuronide respectively. Protein expression and localization were detected by immunoblot and immunohistochemistry. All statistics were non-parametric. RESULTS: UGT, beta-glucuronidase and CYP1A activities were detected in all placentas sampled; CYP2E1 was undetectable. CYP1A, UGT1A UGT2B proteins were detected in all placentas (n = 6) tested and CYP2E1 in 4/6 placentas sampled and were localized to the syncytium. UGT and CYP1A activities were significantly elevated in the placentas of mothers who smoked (P < 0.05 and P < 0.001 respectively) and were greatest in women who both smoked and drank alcohol (P < 0.05 and P < 0.01 respectively). Enzyme activities were significantly negatively correlated with gestational age (P < 0.05, r = 0.54, UGT) and maternal age respectively (P < 0.001, r = 0.63, CYP1A). beta-Glucuronidase activity did not differ with patient variables. CONCLUSIONS: Metabolism of compounds by the human placenta in the first trimester may be affected by maternal and environmental factors altering the activity of constitutive metabolizing enzymes.

Rapid biotransformation of satraplatin by human red blood cells in vitro.

PURPOSE: Satraplatin is an orally administered platinum complex that has demonstrated clinical activity and manageable toxicity in phase II trials. The presence of several different platinum-containing species and very little intact parent drug in the systemic circulation indicates extensive biotransformation of satraplatin in vivo. To investigate the basis for the biotransformation of satraplatin, studies were carried out into the stability of the drug in whole blood and various other biological fluids in vitro. METHODS: Concentrations of satraplatin and platinum-containing biotransformation products in incubation fluids were measured using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS). The fate of satraplatin-derived platinum in whole blood in vitro was determined by analysis of blood fractions for platinum by ICPMS. RESULTS: In fresh human whole blood in vitro, satraplatin concentrations fell very rapidly, resulting in a half-life for the disappearance of the drug of only 6.3 min (95% CI, 5.9 to 6.7 min). After the addition of drug to red blood cells that had been prepared from whole blood and suspended in 0.9% NaCl, satraplatin also disappeared very rapidly. Satraplatin was much more stable in fresh human plasma (t(1/2) 5.3 h) and fully supplemented cell culture medium (t(1/2) 22 h). Two new platinum-containing species appeared on HPLC-ICPMS platinum chromatograms of methanol extracts of plasma after the addition of the drug to whole blood. Their identities were assigned as the platinum(II) complex known as JM118 and a platinated protein with similar electrophoretic mobility to that of serum albumin. During the incubation of satraplatin in blood, platinum associated with red blood cells at an accumulation half-life of 9.5 min (95% CI, 7.1 to 14.2 min). At equilibrium, 62% of the added platinum was associated with red blood cells in a form that was not exchangeable in methanol or 0.9% NaCl. CONCLUSIONS: The rapid disappearance of satraplatin from human blood in vitro depends upon the presence of red blood cells. Generation of JM118 and irreversibly bound membrane- and protein-associated platinum indicates that satraplatin undergoes rapid biotransformation in whole blood.

Thalidomide in cancer treatment: a potential role in the elderly?

There is increased interest in the treatment of cancer with thalidomide because of its antiangiogenic, immunomodulating and sedative effects. In animal models, the antitumour activity of thalidomide is dependent on the species, route of administration and coadministration of other drugs. For example, thalidomide has shown antitumour effects as a single agent in rabbits, but not in mice. In addition, the antitumour effects of the conventional cytotoxic drug cyclophosphamide and the tumour necrosis factor inducer 5,6-dimethylxanthenone-4-acetic acid (DMXAA) were found to be potentiated by thalidomide in mice bearing colon 38 adenocarcinoma tumours. Further studies have revealed that thalidomide upregulates intratumoral production of tumour necrosis factor-alpha 10-fold over that induced by DMXAA alone. Coadministration of thalidomide also significantly reduced the plasma clearance of DMXAA and cyclophosphamide. All these effects of thalidomide may contribute to the enhanced antitumour activity. Recent clinical trials of thalidomide have indicated that it has minimal anticancer activity for most patients with solid tumours when used as a single agent, although it was well tolerated. However, improved responses have been reported in patients with multiple myeloma. Palliative effects of thalidomide on cancer-related symptoms have also been observed, especially for geriatric patients with prostate cancer. Thalidomide also eliminates the dose-limiting gastrointestinal toxic effects of irinotecan. There is preliminary evidence indicating that the clearance of thalidomide may be reduced in the elderly. The exact role of thalidomide in the treatment of cancer and cancer cachexia in the elderly remains to be elucidated. However, it may have some value as part of a multimodality anticancer therapy, rather than as a single agent.

Gender differences in the metabolism and pharmacokinetics of the experimental anticancer agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

PURPOSES: Marked gender differences in the pharmacokinetics of many drugs have been reported. For the investigational anticancer drug, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), negligible gender differences in the plasma pharmacokinetics have been observed in mice. The gender effects on the plasma pharmacokinetics of DMXAA were further investigated using the rat model. In addition, the in vitro metabolism and plasma protein binding of DMXAA in male and female mice, rats and humans were investigated. METHODS: DMXAA was administered to male and female rats by intravenous injection. DMXAA and its major metabolites formed in liver microsomes were determined by HPLC. Unbound DMXAA in plasma was separated by ultrafiltration followed by HPLC determination. RESULTS: In vivo kinetic studies indicated that female rats had 60%, 55% and 73% higher area under the plasma concentration-time curve (AUC) of DMXAA (2413 +/- 188 vs 1505 +/- 312 microM x h, P<0.05), elimination half-life (2.40 +/- 0.45 vs 1.55 +/- 0.33 h) and maximal plasma concentration (Cmax) (1236 +/- 569 vs 716 +/- 280 micro M), but 61% lower plasma clearance than male rats. In vitro studies indicated that male rats had a 67% higher glucuronidation activity (0.75 +/- 0.03 nmol/min per mg) than female rats (0.45 +/- 0.01 nmol/min per mg), resulting in a 96% faster intrinsic clearance (CL(int)) in the males than the females (6.36 +/- 0.65 vs 3.24 +/- .42 ml/min per g, P< 0.05). In contrast, female rats had 25% higher 6-methylhydroxylation activity (0.045 +/- 0.003 nmol/min per mg) than male rats (0.036 +/- 0.002 nmol/min per mg), resulting in a 57% faster intrinsic clearance (CL(int)) in the females than males (0.36 +/- 0.06 vs 0.23 +/- 0.05 ml/min per g). Overall, total CL(int) by both glucuronidation and 6-methylhydroxylation in male rats was 83% higher than in female rats (6.59 +/- 2.11 vs 3.60 +/- 1.07 nmol/min per g). Men ( n=4) had a significantly lower ( P<0.05) CL(int) for glucuronidation than women ( n=10), but a higher CL(int) for 6-methylhydroxylation, resulting in significantly higher total CL(int) in women than men (5.63 vs 8.33 nmol/min per g). There was no significant difference in either the total plasma protein or albumin concentration between male and female mice, rats or humans. CONCLUSION: There were significant gender-related differences in the metabolism and pharmacokinetics in the rat, in contrast to the mouse. This indicates a limited usefulness of the rat as a model for the study of DMXAA metabolism in relation to gender differences, although the gender differences in the in vitro metabolic capacity for DMXAA may provide an explanation for the gender differences in the pharmacokinetics in rats. Data from human liver microsomes may allow the prediction of gender effects in the in vivo pharmacokinetics of DMXAA.