Monitoring Cyp2b10 mRNA expression at cessation of 2-year carcinogenesis bioassay in mouse liver provides evidence for a carcinogenic mechanism devoid of human relevance: the dalcetrapib experience.

INTRODUCTION: Dalcetrapib is a cholesteryl ester transfer protein (CETP) modulator in clinical assessment for cardiovascular outcome benefits. In compliance with regulatory requirements, dalcetrapib was evaluated in rodent 2-year carcinogenesis bioassays. In the mouse bioassay, male mice demonstrated increased liver weight and statistically increased incidences of hepatocellular adenoma/carcinoma. Hepatic cytochrome p450 (Cyp) 2b10 mRNA induction and increased Cyp2b10 enzyme activity signify activation of hepatic nuclear receptor constitutive androstane receptor (CAR), a widely established promoter of rodent-specific hepatic tumors. We therefore monitored hepatic Cyp2b10 mRNA and its enzyme activity in a subset of dalcetrapib-treated male mice from the bioassay. METHODS: Liver samples were obtained from ~1/3 of male mice from each dose group including vehicle-controls (mean and earliest study day of death 678 and 459 respectively). Quantitative real time PCR (qRT-PCR) was performed to determine Cyp2b10 mRNA expression and Cyp1a-, Cyp2b10- and Cyp3a-selective activities were monitored. RESULTS: Cyp2b10 mRNA was strongly induced by dalcetrapib with an expected wide inter-individual variation (5-1421-fold). Group average fold-induction versus vehicle-controls showed a dose-related increase from 48-fold (250mg/kg/day) to 160-fold (750mg/kg/day), which declined slightly at 2000mg/kg/day (97-fold). Cyp enzyme activities showed approximate doubling of total Cyp P450 content per milligram protein and a 9-fold increase in Cyp2b10-selective pentoxyresorufin O-dealkylase activity (750mg/kg/day). DISCUSSION: These data from hepatic Cyp2b10 monitoring are strongly suggestive of CAR activation by dalcetrapib, a mechanism devoid of relevance towards hepatocarcinogenesis in humans; results show feasibility of Cyp2b10 as a surrogate marker for this mechanism at cessation of a carcinogenesis bioassay.

Dalcetrapib: no off-target toxicity on blood pressure or on genes related to the renin-angiotensin-aldosterone system in rats.

BACKGROUND AND PURPOSE: The association between torcetrapib and its off-target effects on blood pressure suggested a possible class-specific effect. The effects of dalcetrapib (RO4607381/JTT-705) and torcetrapib on haemodynamics and the renin-angiotensin-aldosterone system (RAAS) were therefore assessed in a rat model. EXPERIMENTAL APPROACH: Arterial pressure (AP) and heart rate were measured by telemetry in normotensive and spontaneously hypertensive rats (SHR) receiving torcetrapib 10, 40 or 80 mg kg(-1) day(-1); dalcetrapib 100, 300 or 500 mg(-1) kg day(-1); or vehicle (placebo) for 5 days. Expression of RAAS genes in adrenal gland, kidney, aorta and lung from normotensive rats following 5 days' treatment with torcetrapib 40 mg kg(-1) day(-1), dalcetrapib 500 mg kg(-1) day(-1) or vehicle was measured by quantitative polymerase chain reaction. KEY RESULTS: Torcetrapib transiently increased mean AP in normotensive rats (+3.7 +/- 0.1 mmHg), whereas treatment in SHR resulted in a dose-dependent and sustained increase [+6.5 +/- 0.6 mmHg with 40 mg kg(-1) day(-1) at day 1 (P < 0.05 versus placebo)], which lasted over the treatment period. No changes in AP or heart rate were observed with dalcetrapib. Torcetrapib, but not dalcetrapib, increased RAAS-related mRNAs in adrenal glands and aortas. CONCLUSIONS AND IMPLICATIONS: In contrast to torcetrapib, dalcetrapib did not increase blood pressure or RAAS-related gene expression in rats, suggesting that the off-target effects of torcetrapib are not a common feature of all compounds acting on cholesteryl ester transfer protein.

[Concentrations of diclofenac in aqueous humor and in plasma after i.v. injection in 59 patients undergoing cataract surgery--a prospective study]. / Konzentrationsverlauf von Diclofenac im Kammerwasser und im Plasma bei i.v. Applikation vor der Kataraktextraktion bei 59 Patienten--eine prospektive Studie.

BACKGROUND: The mean diclofenac level in the aqueous humor after i.v.-injection should be examined as an example for other systemic applied drugs. The clinical effect is to be evaluated with regard to perioperative anti-inflammatory therapy in patients undergoing cataract surgery. PATIENTS AND METHODS: In this prospective clinical study 59 patients (age 41-86 years) scheduled for phacoemulsification by the tunnel technique, received 75 mg diclofenac-sodium by short time i.v.-infusion (30 min) within an interval between 30 min and 7 h before surgery. Aqueous humor samples and one blood sample were taken at the beginning, and a second blood sample was taken at different time intervals before surgery. RESULTS: The plasma concentration of diclofenac one minute after infusion was 13,961 +/- 4277 ng/ml. The concentration decreased rapidly during the distribution phase (1305 +/- 324 ng/ml after 40 min) and reached after 336 min 419 +/- 277 ng/ml. A concentration of 83.9 ng/ml diclofenac was found for the last sampling point after 353 min in the elimination phase. The mean diclofenac concentration in the aqueous humor reached after about 60 min the highest level (21.7 +/- 12.7 ng/ml). This level decreases to 4.4 +/- 0.4 ng/ml (120 min) and 3.1 +/- 1.3 ng/ml (300 min), respectively. CONCLUSIONS: The calculated mean residence time of diclofenac molecules in aqueous humor of 123 min matched the mean disposition residence time of diclofenac in the body (119 min).

Pharmacokinetics and toxicity of idarubicin in the rat.

This study was designed to examine the pharmacokinetics and toxicity of idarubicin (IDA) in rats. In two groups of rats IDA was infused either into the V. iugularis interna or into the A. carotis communis, respectively. The venous plasma concentration of IDA and its primary metabolite idarubicinol (IDOL) were measured up to 48 hours by high-performance liquid chromatography (HPLC) with fluorescence detection. The weights of the rats and the levels of haemoglobin, leukocytes, and thrombocytes were recorded. The plasma concentration-time data were analysed, assuming a biexponential disposition curve, both by the traditional (two-stage) method and by population pharmacokinetic modelling. The basic pharmacokinetic parameters clearance (CL = 27.0 ml min(-1)), mean disposition residence time (MDRT = 519.2 min), and volume of distribution at steady state (Vss = 12.51) were estimated for IDA. The mean residence time (MRT) of the generated IDOL was 2982.5 min. No significant differences between pre- and postpulmonal injection were found in the pharmacokinetics and pharmacodynamics of IDA. The mean survival time of 13.3 days is attributed to a severe myelosuppression.

Cytoplasmic binding and disposition kinetics of diclofenac in the isolated perfused rat liver.

1. The binding kinetics of diclofenac to hepatocellular structures were evaluated in the perfused rat liver using the multiple indicator dilution technique and a stochastic model of organ transit time density. 2. The single-pass, in situ rat liver preparation was perfused with buffer solution (containing 2% albumin) at 30 ml min(-1). Diclofenac and [(14)C]-sucrose (extracellular reference) were injected simultaneously as a bolus dose into the portal vein (six experiments in three rats). An analogous series of experiments was performed with [(14)C]-diclofenac and [(3)H]-sucrose. 3. The diclofenac outflow data were analysed using three models of intracellular distribution kinetics, assuming (1) instantaneous distribution and binding (well-mixed model), (2) 'slow' binding at specific intracellular sites after instantaneous distribution throughout the cytosol (slow binding model), and (3) 'slowing' of cytoplasmic diffusion due to instantaneous binding (slow diffusion model). 4. The slow binding model provided the best description of the data. The rate constants for cellular influx and sequestration were 0.126+/-0. 026 and 0.013+/-0.009 s(-1), respectively. The estimated ratio of cellular initial distribution volume to extracellular volume of 2.82 indicates an almost instantaneous distribution in the cellular water space, while the corresponding ratio of 5.54 estimated for the apparent tissue distribution volume suggests a relatively high hepatocellular binding. The non-instantaneous intracellular equilibration process was characterized by time constants of the binding and unbinding process of 53.8 and 49.5 s, respectively. The single-pass availability of diclofenac was 86%. The results obtained with [(14)C]-diclofenac and [(3)H]-sucrose were not statistically different.

Determination of idarubicin and idarubicinol in rat plasma using reversed-phase high-performance liquid chromatography and fluorescence detection.

A reversed-phase high-performance liquid chromatographic method is described for the simultaneous determination of idarubicin and idarubicinol in rat plasma. Blood samples were analyzed from 16 rats which had received an intravascular dose of 2.25 mg kg(-1) idarubicin. After deproteinization with acetonitrile, the separation was performed with a LiChrospher 100 RP-18 column (5 microm), using fluorescence detection (excitation: 485 nm/emission: 542 nm). The mean recovery was 95.6% for idarubicin and 90.7% for idarubicinol, respectively. The detection limit was 0.25 ng ml(-1) using an injection volume of 50 microl. Daily relative standard deviation (RSD) was 3.2% (10 ng idarubicin/ml, n=10) and 4.4% (10 ng idarubicinol/ml, n=10).

Simultaneous determination of diclofenac and oxybuprocaine in human aqueous humor with HPLC and electrochemical detection.

A sensitive and selective bioanalytical method for simultaneous determination of diclofenac and oxybuprocaine in human aqueous humor using reversed-phase HPLC and electrochemical detection is described. Chromatographic separation was achieved by using a Regis SPS 100 RP-8 column (5 microns; 150 x 4.6 mm I.D.). This support is coated with a hydrophilic polyoxyethylenepolymer. It allows protein-containing samples to be injected directly onto the column. The electrochemical detector permit a detection limit of 500 pg diclofenac per ml (daily relative standard deviation 6.3%) and 50 ng oxybuprocaine per ml (daily R.S.D. 2.6%), respectively. Results of administered and measured drug-concentrations in time dependent decrease are presented.

Crocheted ETFE-reactor for on-line post-column photoderivatization of diclofenac in high-performance liquid chromatography.

A sensitive and selective bioanalytical method for diclofenac using reversed-phase HPLC and fluorescence detection is described. Diclofenac was detected as its fluorescent derivative after on-line post-column photoderivatization. Irradiation with UV light of diclofenac in aqueous solutions leads to the sequential loss of both chlorine substituents and ring closure. The major product, carbazole-1-acetic acid, was detected by a fluorescence detector using an excitation wavelength of 286 nm and an emission wavelength of 360 nm. The self-made reactor was a crocheted ethylene and tetrafluoroethylene (ETFE, named TEFZEL) capillary, 20 m in length, wound directly around a 253.7 nm UV lamp. The capillary was crocheted in order to overcome peak widening. Chromatographic separation was achieved by using a Regis SPS 100 RP-8 column (5 microm; 150 mm x 4.6 mm i.d.) and a LiChrospher 100 RP-18 (5 microm) guard column from E. Merck. The detection limit was 1 ng ml(-1) at an injection volume of 20 microl. Daily relative standard deviations (RSD) were 5.5%, (73 ng diclofenac/ml, n = 9), and 5.1% (405 ng diclofenac/ml, n = 6), respectively. Chromatograms of human aqueous humor and human serum containing diclofenac, and figures showing the time dependent increase/decrease of the photoderivatization product, are shown.