Different sensitivity to nephrotoxic agents and osmotic stress in proximal tubular and collecting duct cell lines derived from transgenic mice

We established six renal tubular cell lines from definite tubular areas of the kidney of transgenic mice harboring tsSV40 large T-antigen gene. Three are proximal tubular cell lines prepared from the S₁, S₂ and S₃ segments of the proximal tubule and the others are collecting duct cell lines obtained from cortical, outer medullary and inner medullary collecting ducts (CCD, OMCD and IMCD, respectively). To verify the growth properties of these cell lines under different temperature conditions (33 and 39 °C), two representative cells were chosen from the proximal tubule (S₁ cells) and from the collecting duct (IMCD cells). From these cells, a daily change in cell number was evaluated as a parameter of cell growth. As might be expected, cell numbers of these cells increased only at 33 °C. Similar patterns were also observed with the other cell lines. To observe the different sensitivity to nephrotoxic agents in proximal tubular cell lines, the cells were exposed to nephrotoxic agent, gentamicin, ochratoxin A or cisplatin. Gentamicin (1 mg/ml) dose-dependently decreased cellular ATP levels of the S₁ cells only. In contrast, the effect of ochratoxin A (10⁻⁶ m) was most pronounced in the S₂ cells, and that of cisplatin (10 μg/ml) in the S₃ cells. To characterize collecting duct cell lines, a hyperosmotic challenge of 700 or 1100 mOsm/l was applied to the cells. At an isoosmotic condition of 300 mOsm/l, the number of cells from the collecting ducts, regardless of their origin, increased continuously during the culture period of 4 days. At an osmotic concentration of 700 mOsm/l, the number of CCD cells decreased, while OMCD cells showed a gradual but a significant increase in cell numbers throughout the culture period. IMCD cells, however, proliferated even at a concentration as high as 1100 mOsm/l, although an initial decrease in cell number was noted on the first day of culture. For confirmation of intracellular free calcium ([Ca²⁺]_i) mobilization, cells were treated with ATP and bradykinin. The [Ca²⁺]_i was increased significantly and immediately by ATP (10⁻⁴ m) in S₁ cells and bradykinin (10⁻⁷ m) in IMCD cells. From the results obtained, it is indicated that renal tubular cell lines from transgenic mice have different sensitivities to nephrotoxic or osmotic stress showing the conservation of the functional characters of the definite part it originated from.     

Effects of paraquat,dinoseb and 2,4-D on intracellular calcium and on vasopressin-induced calcium mobilization in isolated hepatocytes

 The effects of the herbicides paraquat, dinoseb and 2,4-D on intracellular Ca²⁺ levels and on vasopressin-induced Ca²⁺ mobilization were investigated in intact isolated hepatocytes. Incubation of rat hepatocytes with paraquat (5 mM for 60 min) and dinoseb (10 μM) resulted in a time-dependent loss of viability by approximately 25%. Viability of cells treated with 2,4-D decreased significantly, dropping to about 20% at 10 mM and 60 min incubation. Exposure of hepatocytes to paraquat (1–10 mM) for 60 min had no effect on the basal level of [Ca²⁺] _i . Additionally, exposure to paraquat had no effect on the magnitude and on the duration of the [Ca²⁺] _i response to vasopressin. In the presence of 2,4-D (1–10 mM), basal [Ca²⁺] _i increases as a function of herbicide concentration. The magnitude of the Δ[Ca²⁺] _i response decreases from 256±8 nM in control to 220±5 nM, at 10 mM 2,4-D. Exposure of hepatocytes to dinoseb (1–10 μM) had no effect on the basal level of [Ca²⁺] _i . However, a strong concentration-dependent decrease in the magnitude of Δ[Ca²⁺] _i in response to vasopressin was noticed at 60 min incubation. Dinoseb markedly inhibited the stimulation of the production of inositol phosphates by vasopressin stimulus. The present study demonstrates that paraquat, 2,4-D and dinoseb cause cell death in hepatocytes by mechanisms not related to an early increase in [Ca²⁺] _i . Additionally, it has been shown for the first time that dinoseb disturbs the transduction mechanism promoted by vasopressin by inhibiting the formation of IP₃. Received: 11 October 1994/Accepted: 5 December 1994     

Effect of bolesatine on phospholipid/calcium dependent protein kinase in vero cells and in rat thymus

 Bolesatine, a glycoprotein from Boletus satanas Lenz, has previously been shown to be mitogenic to rat and human lymphocytes at very low concentrations, whereas higher concentrations inhibit protein synthesis in vitro and in several in vivo systems. The mechanism whereby this mitogenic activity occurs was previously unknown. To elucidate this mechanism, the effects of bolesatine have been studied in a cell-free system, VERO cells in culture, and in rat thymus. Bolesatine was found to activate PKC, in vitro (cell free system), in VERO cells, and in vivo in rat thymus. In a cell-free system, bolesatine appears to be a direct effector of PKC. The activation is concentration dependent for 1 – 10 ng/ml. At the same time, VERO cells significantly proliferate when incubated with bolesatine (3, 5 and 10 ng/ml), since the DNA synthesis increases by 27, 48 and 59%, for respectively, 3, 5 and 10 ng/ml compared with control. Moreover, Bolesatine (5 and 10 ng/ml) induces InsP₃ release in a concentration-dependent manner (114 and 142%) as compared to control. In vivo, 24 h after oral administration of bolesatine to rats (20, 100 and 200 μg/kg), PKC activity is significantly increased in thymus. The most effective doses (100 and 200 μg/kg) give 590 – 620% increase in cytosolic PKC activity and 85 – 91% increase in total PKC activity as compared to control. This PKC activation by bolesatine in rat thymus is directly linked to the mitogenic activity observed in vivo. Bolesatine is thus capable of activating the PKC directly and/or indirectly (via InsP₃ release) during its mitogenic process. Received: 6 February 1995 / Accepted: 20 March 1995     

Pharmacokinetics and tolerability of formoterol in healthy volunteers after a single high dose of Foradil dry powder Inhalation via aerolizerTM

Objective: The pharmacokinetics of the long-acting β₂-agonist formoterol fumarate, which is a racemate of the (S,S)- and (R,R)-enantiomers were evaluated in 12 healthy (eight male, four female) volunteers after a single inhaled high dose of 120 μg of formoterol fumarate. The tolerability and safety were also assessed. Methods: Each volunteer inhaled the single 120-μg dose through the Aerolizer device within 2–5 min, using ten 12-μg dry powder capsules for inhalation. Formoterol, i.e., the sum of both enantiomers, was determined in plasma over 24 h, whereas the separate enantiomers were determined in urine over 48 h. Incidence, seriousness and severity of adverse experiences, electrocardiogram (ECG), including the corrected QT interval (QTc) calculation, systolic blood pressure, heart rate, and plasma potassium levels were recorded. Results: In nine of the 12 volunteers, the peak plasma concentration of formoterol was observed already at 5 min after inhalation. The absorption kinetics were complex, as depicted by multiple peaks or shoulders within 0.5–6 h after inhalation. Mean with (SD; n = 12) of maximum concentration (C_max) and area under the curve (AUC) of formoterol in plasma were 266 (108) pmol · l⁻¹ and 1330 (398) pmol · h · l⁻¹, respectively. The moderate inter-individual variability in systemic exposure of formoterol reflects the homogeneous pharmacokinetics of the drug. A predominant slow elimination of formoterol from plasma with a mean half-life (t_1/2) of 10 h was demonstrated. Assuming linear kinetics in plasma suggested by urinary data, the steady-state trough plasma levels of formoterol for a b.i.d. dosing regimen are predicted to amount to 20% of C_max. In urine, mean with (SD; n = 10) of the amount excreted over 48 h was 3.61 (0.89)% of dose for the pharmacologically active (R,R)-enantiomer and 4.80 (1.33)% of dose for the (S,S)-enantiomer. The terminal half-lives calculated from the excretion rate-time curves, i.e., 13.9 h and 12.3 h for the (R,R)- and (S,S)-enantiomer, respectively, confirm the slow elimination of formoterol from plasma. The dose inhaled was 10 times the most frequently recommended dose (12 μg) and 5 times the highest recommended dose (24 μg). Ten of 12 subjects experienced mild and transient nervousness. Pulse readings demonstrated the maximum mean increase of 25.8 beats · min⁻¹ at 6 h. The mean maximum QTc increase was 25 msec at 6 h. Pulse and QTc values returned to baseline or close to baseline values at 24 h or before. Potassium levels in plasma decreased in eight out of 12 subjects; the lowest mean value was 3.53 mmol · l⁻¹ at 2 h post-dose. The lowest individual potassium measurement was 2.95 mmol · l⁻¹ between 15 min and 6 h. By 8 h post-dose all values had returned to within the normal ranges. Conclusions: The extremely fast appearance of formoterol in plasma shows the predominance of airways absorption shortly after inhalation. Due to a terminal elimination half-life of about 10 h, sustained systemic concentrations of formoterol are predicted for a twice daily treatment regimen without noteworthy accumulation. The excreted amounts in percent of dose of the enantiomers in urine and the enantiomer ratio are similar to data reported previously after lower doses and suggest linear kinetics for doses between 12 μg and 120 μg of formoterol fumarate. The expected side effects on heart rate, QTc interval, and plasma potassium were small and had no clinical consequences in spite of the very high dose of 120 μg (5 to 10 times the recommended therapeutic dose of Foradil). It should be noted that the impact of high doses may be greater in patients. Nevertheless these findings provide reassurance on the safety margin of formoterol after accidental and intentional overdosing. Received: 22 June 1998 / Accepted in revised form: 2 December 1998     

Effect of DMPS and various adsorbents on the arsenic excretion in guinea-pigs after injection with As2O3

The present experiments were performed to test the possibility of interrupting the enterohepatic circulation of arsenic (As). Therefore the efficacy of adsorbents to bind As and/or As-DMPS adducts in vitro and their effect on the excretion of As into the feces and urine in vivo were investigated after injection of As₂O₃ and DMPS in guinea-pigs. The adsorbents bentonite, activated charcoal or colestyramine, respectively, were tested. Only slight binding of ⁷³As (<5% of the ⁷³As dose) was observed to all adsorbents in vitro. After addition of DMPS, a good binding was found for ⁷³As to colestyramine (50%) or activated charcoal (60%), respectively. However, the ⁷³As-DMPS adduct was removed from the activated charcoal during washing. In the first in vivo experiment, male guinea-pigs (n=4/group) received As₂O₃ [0.02 mmol As(III)/kg s.c. labelled with a tracer dose of ⁷³As(III) (0.14 kBq/g)], 30 min later DMPS (0.1 mmol/kg i.p.) and by gastric tube (10 ml/kg body wt) either saline, bentonite (1 g/kg), activated charcoal (1 g/kg) or colestyramine (0.2 g/kg), respectively. Urine and feces were collected for 24 h. No increase in ⁷³As excretion into the feces was observed after administration of DMPS and all adsorbents, compared to control animals. In the second in vivo experiment male guinea-pigs (n=4/group) received the same As₂O₃ (+⁷³As)-and DMPS dose. In addition, with a gastric tube (10 ml/kg) saline, colestyramine (0.2 g/kg), DMPS (0.1 mmol/kg), or the combination of DMPS (0.1 mmol/kg) + colestyramine (0.2 g/kg) were administered according to the scheme given in the following table. The amount of feces excreted did not differ between groups. Excretion of ⁷³As within the feces during the first 12 h after As injection is shown in the following table (mean±SEM). The same amount of ⁷³As (34 % of the ⁷³As dose) was excreted into the urine from animals in groups 4 and 5 during this time. Obviously, the combined oral administration of DMPS+colestyramine markedly enhanced fecal excretion of As mobilized by DMPS i.p. It is suggested that interruption of enterohepatic circulation of As may be a valuable adjunct in the treatment of As poisoning. Received: 10 January 1995/Accepted: 6 March 1995     

Effect of lead on tube formation by cultured human vascular endothelial cells

 The effect of lead acetate (Pb) on the growth of and tube formation by cultured human umbilical vascular endothelial cells (HUVEC) was examined. HUVEC were collected by enzymatic digestion with collagenase. The number of viable cells of HUVEC was negligibly affected by cultivation with Pb at concentrations of 1 – 100 μM, but was slightly reduced by cultivation at 500 μM. Tube formation was studied by culturing the cells on a gelled basement membrane matrix (Matrigel). Treatment of HUVEC with 0.1 – 50.0 μM Pb for 24 h inhibited tube formation dose-dependently. The length of tube formation decreased time-dependently with 1.0 μM Pb. These findings suggest that Pb inhibits the formation of a capillary network by HUVEC, and that Pb could be injurious to endothelial cell function. Received: 23 January 1995 / Accepted: 20 March 1995     

Modulation of energy status and cytotoxicity induced by FK506 and cyclosporin A in a renal epithelial cell line

FK506 and cyclosporin A (CsA) are two potent immunosupressants with similar toxicity profile. Nephrotoxicity is the main adverse effect of both compounds. The aim of this study is to compare the in vitro nephrotoxic effects on renal epithelial cell line LLC-PK1 by measuring cell viability and energy status as evaluated by concentrations of ATP and ATP metabolites. Cell viability (expressed as IC₅₀ was assessed via thiazolyl blue (MTT) assay after incubation for 4–24 h with FK506 or CsA. ATP and its metabolites were determined by HPLC after 4 and 6 h incubation with FK506 or CsA alone at the respective IC₅₀. Both FK506 and CsA decreased cell viability to similar extents, in a dose- and time-dependent manner. After 4 h incubation, both drugs decreased ATP levels (−25%) and increased uric acid levels. However, the latter percentage increase was twofold higher with CsA (18%) than with FK506 (9%). The energy charge, calculated according to levels of adenine nucleotides, was decreased by 10% in FK506-treated cells and by 27% in CsA-treated cells. At the end of 6-h incubation, FK506-treated cells maintained ATP levels coupled with energy charge at near control levels whereas the levels were 32% lower in CsA treated cells. Compared to the 4 h-incubation, the increase in uric acid was similar for FK506 but was doubled with CsA. The decrease in cell integrity and ATP depletion induced by CsA in LLC-PK1 cells was only transiently observed with FK506. By preserving energy status, FK506 leads to fewer metabolic disturbances than CsA in the renal epithelial cell line LLC-PK1, demonstrating a minor potential nephrotoxicity. Received: 8 January 1997 / Accepted: 6 March 1997     

Population analysis of the non-linear red blood cell partitioning of draflazine following various infusion durations

Objective: The pharmacokinetics and non-linear red blood cell partitioning of the nucleoside transport inhibitor draflazine were investigated in 19 healthy male and female subjects (age range 22–55 years) after a 15-min i.v. infusion of 1 mg, immediately followed by infusions of variable rates (0.25, 0.5 and 1 mg · h⁻¹) and variable duration (2–24 h). Methods: The parameters describing the capacity-limited specific binding of draflazine to the nucleoside transporters located on erythrocytes were determined by NONMEM analysis. The red blood cell nucleoside transporter occupancy of draflazine (RBC occupancy) was evaluated as a pharmacodynamic endpoint. Results: The population typical value for the dissociation constant K _d (%CV) was 0.648 (12) ng · ml⁻¹ plasma, expressing the very high affinity of draflazine for the erythrocytes. The typical value of the specific maximal binding capacity B_max (%CV) was 155 (2) ng · ml⁻¹ RBC. The interindividual variability (%CV) was moderate for K _d (38.9%) and low for B_max (7.8%). As a consequence, the variability in RBC occupancy of draflazine was relatively low, allowing the justification of only one infusion scheme for all subjects. The specific binding of draflazine to the red blood cells was a source of non-linearity in draflazine pharmacokinetics. Steady-state plasma concentrations of draflazine virtually increased dose-proportionally and steady state was reached at about 18 h after the start of the continuous infusion. The t_1/2βaveraged 11.0–30.5 h and the mean CL from the plasma was 327 to 465 ml · min⁻¹. The disposition of draflazine in whole blood was different from that in plasma. The mean t_1/2β was 30.2 to 42.2 h and the blood CL averaged 17.4–35.6 ml · min⁻¹. Conclusion: Although the pharmacokinetics of draflazine were non-linear, the data of the present study demonstrate that draflazine might be administered as a continuous infusion over a longer time period (e.g., 24 h). During a 15-min i.v. infusion of 1 mg, followed by an infusion of 1 mg · h⁻¹, the RBC occupancy of draflazine was 96% or more. As the favored RBC occupancy should be almost complete, this dose regimen could be justified in patients. Received: 6 February 1997 / Accepted in revised form: 12 May 1997     

Lack of pharmacokinetic interaction between ropinirole and theophylline in patients with Parkinson's disease

Objective: Ropinirole and theophylline have the potential to interact, because they use the same hepatic cytochrome P450 (CYP1A2) as their major metabolic pathway. The present study investigated the effect of steady-state oral theophylline on the pharmacokinetics of ropinirole at steady state and the effect of steady-state ropinirole on the pharmacokinetics of a single intravenous (i.v.) dose of theophylline, both in patients with idiopathic Parkinson's disease (PD). Methods: Pharmacokinetic parameters (AUC and C_max) for i.v. theophylline were compared before and after a 4-week period of oral treatment with ropinirole (2 mg t.i.d.) in 12 patients with PD. Patients were then maintained at this dose of ropinirole, and oral theophylline was co-administered at doses of up to 300 mg b.i.d. The parameters AUC, C_max and t_max for ropinirole were compared before, during and after oral theophylline co-treatment. Results: Co-administration of ropinirole did not significantly change the pharmacokinetics of i.v. theophylline (mean AUC with and without ropinirole: 68.6 μg · h⁻¹ · ml⁻¹ and 70.0 μ· h⁻¹ · ml⁻¹, respectively; mean C_max with and without ropinirole: 11.07 μ g · ml⁻¹ and 11.83 μg · ml⁻¹, respectively). Similarly, there were no significant changes in ropinirole pharmacokinetics when the drug was co-administered with oral theophylline (mean AUC for ropinirole with and without theophylline: 21.91 ng · h⁻¹ · ml⁻¹ and 22.09 ng · h⁻¹ · ml⁻¹, respectively; mean C_max for ropinirole with and without theophylline: 5.65 ng · ml⁻¹ and 5.54 ng · ml⁻¹, respectively; median t_max for ropinirole with and without theophylline: 2.0 h and 1.5 h, respectively). Conclusion: These results suggest a lack of significant pharmacokinetic interaction between the two drugs at current therapeutic doses. Received: 10 August 1998 / Accepted in revised form: 27 January 1999     

Lack of a pharmacokinetic interaction between atovaquone and proguanil

Objective: To assess the magnitude of the putative effect of atovaquone on the pharmacokinetics of proguanil and to determine whether the pharmacokinetics of atovaquone are affected by concomitant administration of proguanil, with both drugs administered for 3 days to healthy adult volunteers. Methods: This was an open-label, randomized, three-way cross-over study, in which 18 healthy volunteers received 400 mg proguanil, 1000 mg atovaquone and 1000 mg atovaquone + 400 mg proguanil. Each treatment was given once daily for 3 days with a 3-week wash-out period between each occasion. For the assay of proguanil, cycloguanil and atovaquone, blood was sampled before dosing and at regular intervals over 8 days when proguanil was given, and over 17 days when atovaquone was given. Results: The geometric mean of the area under the atovaquone plasma concentration-time curve calculated from 0 to 24 h after the last dose (AUC_0→24h) was 180 μg · ml⁻¹ · h following administration of atovaquone alone and 193 μg · ml⁻¹ · h following atovaquone with proguanil. The geometric mean AUC_0→24h for proguanil was 6296 ng · ml⁻¹ · h after proguanil alone and 5819 ng · ml⁻¹ · h following proguanil with atovaquone. The corresponding values for the metabolite cycloguanil were 1297 ng · ml⁻¹ · h and 1187 ng · ml⁻¹ · h, respectively. The geometric mean elimination half-life (t_1/2) of atovaquone was 57.1 h when given alone and 59.0 h when administered together with proguanil. The corresponding geometric mean values of t_1/2 for proguanil were 13.7 h and 14.5 h. Exploratory statistical analysis showed no important gender effects on the pharmacokinetics of atovaquone, proguanil, or cycloguanil. Conclusion: The pharmacokinetics of atovaquone and proguanil and its metabolite, cycloguanil, were not different when atovaquone and proguanil were given alone or in combination. Received: 14 October 1998 / Accepted in revised form: 8 February 1999     

Cardiotoxicity of adrenochrome in isolated rabbit hearts assessed by epicardial NADH fluorescence

Noradrenaline in a micromolar concentration has recently been shown to contribute to ischemic tissue injury by direct cardiotoxic effects independent of functional alterations. Oxygen free radicals, generated during the auto-oxidation of catecholamines, are important mediators of catecholamine cardiotoxicity. However, the role of the oxidative products (aminochromes) is still unclear. We examined the effects of adrenochrome on functional parameters and on regional myocardial ischemia (MI) in isolated electrically-driven rabbit hearts with depleted catecholamine stores (reserpine 7.0 mg/kg i.p. 16 – 24 h before preparation, Langendorff, constant pressure: 70 cm H₂O, Tyrode solution, Ca⁺⁺ 1.8 mmol/l, 37°C). Repetitive MI, separated by a reperfusion period of 50 min, was induced by coronary artery branch ligature, and MI was quantitated from epicardial NADH fluorescence photography. Adrenochrome-treatment (10^– 6 M or 10^– 4 M) was started after a reperfusion period of 20 min. The left ventricular pressure (LVP) was significantly enhanced by adrenochrome (p <0.05), but it fell thereafter to below its initial value in hearts treated with adrenochrome 10^– 4 M. The global coronary flow (CF) was not affected by adrenochrome 10^– 6 M (P >0.05), but it was significantly decreased by adrenochrome 10^– 4 M (P <0.05). The relative CF (= CF/LVP × heart-rate) was numerically decreased by adrenochrome 10^– 6 M (p >0.05) and more markedly by adrenochrome 10^– 4 M (p <0.05). Whereas epicardial NADH fluorescence was similar after repetitive coronary artery occlusions in controls and in hearts treated with adrenochrome 10^– 6 M (p >0.05), it was significantly enhanced by adrenochrome 10^– 4 M (p <0.05). In isolated rabbit hearts, adrenochrome possesses deleterious effects on MI only at a very high concentration but not in a micromolar concentration. Therefore, it seems that aminochromes may be less cardiotoxic than catecholamines. Received: 28 February 1994 / Accepted: 2 May 1994     

Adrenocortical activity with repeated administration of one-daily inhaled fluticasone propionate and budesonide in asthmatic adults

Objective: The aim of this study was to evaluate the steady-state effects of once-daily inhaled fluticasone propionate (FP) and budesonide (BUD) on adrenocortical activity in asthmatic patients. Methods: Ten asthmatic patients with a mean age of 31.2 years, a mean forced expiratory volume in 1 s (FEV₁) of 91% predicted and a forced mid-expiratory flow (FEF_25–75) of 62.3% predicted were studied in a single-blind randomised crossover design comparing placebo (PL), FP (375 μg per day and 750 μg per day) and BUD (400 μg per day and 800 μg per day) all given once daily for 4 days at each dose via a pressurised metered dose inhaler (pMDI) at 0800 hours. After 4 days of treatment, plasma cortisol was measured at 0800 hours (24 h after the last dose) and a 10-h overnight urine collection was taken, 14 h after the last dose (2200–0800 hours) for analysis of cortisol and creatinine excretion. Results: Plasma cortisol levels (nmol · l⁻¹, as geometric mean) at 0800 hours demonstrated a significant difference between the highest doses of FP and BUD (424.1 vs 510.3 nmol · l⁻¹, respectively) but not between the low doses (506.8 vs 514.9 nmol · l⁻¹; PL 532.2 nmol · l⁻¹). For the highest dose FP (750 μg) this equated to 20% suppression of 0800 hours plasma cortisol. Likewise, for overnight urinary cortisol output (nmol · 10 h⁻¹, as geometric mean), there was a significant difference at the high doses of FP and BUD (25.5 vs 38.2 nmol · 10 h⁻¹), but not at the low doses 31.3 vs 34.8 nmol · 10 h⁻¹; PL 32.0 nmol · 10 h⁻¹. For the overnight urinary cortisol/creatinine ratio (nmol · mmol⁻¹, as geometric mean) there was a similar trend; 4.5 vs 6.1 nmol · mmol⁻¹ for high dose and 5.6 vs 6.3 nmol · mmol⁻¹ for low dose; PL 5.9 nmol · mmol⁻¹. Conclusion: Repeated doses of FP 750 μg once daily caused greater adrenal suppression than BUD 800 μg once daily, when comparing effects on plasma cortisol levels at 0800 hours, 24 h after the last dose, as well as effects on overnight urinary cortisol output. Neither FP 375 μg once daily nor BUD 400 μg once daily produced detectable adrenal suppression. Received: 29 April 1997 / Accepted in revised form: 5 July 1997     

Pharmacokinetics of oxypolygelatine in healthy volunteers

Objective/methods: The pharmacokinetics of the plasma substitute oxypolygelatine (OPG) were studied in 12 healthy volunteers after single-dose administration of 27 ml · kg⁻¹ body weight, with a maximum of 2000 ml. OPG was determined in plasma and urine over 48 h after the infusion. Peak plasma OPG concentrations at the end of the infusion were determined to 4.600 (623) μg · ml⁻¹, the area under the plasma concentration/time curve (AUC_0∞) was calculated to 70.135 (15.861) μg · h · ml⁻¹. Results: The model-independently calculated volume of distribution came to 23.1 (4.8) l with a clearance total is (Cl_tot) of 24.6 (6.8) ml · min⁻¹. The initial half-life according to a three-compartment model came to 0.3 (0.2) h, followed by a distribution half-life of 3.1 (2.6) h and a terminal elimination half-life of 13.4 (2.2) h. Cumulative urinary excretion of OPG was 64% after 48 h. Conclusion: This low recovery rate may be explained by the distribution of OPG into the extravascular space and subsequent degradation in tissue sites. Received: 9 June 1998 / Accepted in revised form: 23 November 1998     

Inhibitory activity of Bifidobacterium adolescent combined with cisplatin on melanoma in mice and its mechanism

The aim of this study is to explore inhibitory activity of Bifidobacterium adolescent combined with cisplatin on the growth of melanoma (B16) in mice and the underlying mechanism. C57 mice were inoculated with B16 cancer cells to construct mouse model of melanoma and treated with bifidobacterium adolescent combined with cisplatin. Ratios of inhibitory activity on the growth of melanoma (B16) were calculated. Pathology changes of the tumor were observed by HE staining. B16 cell cycles were examined on a flow cytometer. Lymphocyte proliferation was measured with MTT assay and the T-cell subset was measured by double marked fluorescence. When bifidobacterium of 10¹⁰ cfu/L was injected, the ratio of inhibitory activity on the growth of melanoma (B16) reached 54%, which was similar to that of cisplatin group. The ratio of inhibitory activity reached 74.45% when the mice were treated by bifidobacterium combined with cisplatin. HE staining shows that bifidobacterium inhibited B16 cell proliferation and enhanced the cisplatins killing activity on B16 cells. The results of flow cytometry demonstrated that B16 cell proliferation was arrested at G₁ stage after treatment with bifidobacterium. The B16 cell proliferation was arrested at S stage after treatment with cisplatin. The CD4+ percentage increased and the difference was significant compared with the normal group after treatment with bifidobacterium, indicating that T-cell immune activity was enhanced. Treatment with bifidobacterium combined with cisplatin can enhance the inhibitory activity on the growth of melanoma (B16) of cisplatin. The mechanism of the inhibitory activity on B16 cell proliferation is correlated with the enhanced immune activity in mice. __________ Translated from Journal of the Fouth Military Medical University, 2007, 28(21): 1947–1950 [译自: 第四军医大学学报]     

Food increases the bioavailability of mefloquine

Objectives: To assess the effect of food on the pharmacokinetics of the antimalarial mefloquine and its major plasma metabolite in healthy volunteers. Methods: In an open, two-way cross-over study, 20 healthy male volunteers who had fasted overnight were randomised to receive a single oral dose of 750 mg mefloquine in the absence or presence of a standardised, high-fat breakfast, administered 30 min before drug administration. Blood samples were taken at specific times over an 8-week period. Plasma concentrations of mefloquine and its carboxylic acid metabolite were determined by high-performance liquid chromatography for pharmacokinetic evaluation. Results: The parameters C_max and AUC of both mefloquine and its metabolite were significantly (P < 0.05) higher under post-prandial conditions than under fasting conditions (mefloquine: mean C_max 1500 vs 868 μg · l⁻¹, mean AUC 645 vs 461 mg l⁻¹ · h; metabolite: C_max 1662 vs 1231 μg · l⁻¹, AUC 1740 vs 1310 mg l⁻¹ · h). The intersubject variability in C_max and AUC of mefloquine was less than 30% (coefficient of variation). The time to peak plasma concentration of mefloquine was significantly shorter after food intake (17 vs 36 h). Compared with absorption in volunteers who had fasted, food did not alter t_1/2 (mefloquine and its metabolite) and t_max (metabolite). Conclusion: Under the conditions of this study, food increases the rate and the extent of mefloquine absorption. It is reasonable to recommend that mefloquine be administered with food in travellers receiving chemoprophylaxis and in patients on recovery receiving curative treatment. In acutely ill patients, mefloquine should be taken as soon as possible and administration with or shortly after meals should be attempted as soon as feasible. Received: 10 February 1997 / Accepted in revised form: 16 June 1997     

Determination of the relative bioavailability of nedocromil sodium to the lung following inhalation using urinary excretion

Objective: To determine the effects of cimetidine on the steady-state pharmacokinetics and pharmacodynamics of atorvastatin, a 3-hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. Methods: Twelve healthy subjects participated in a randomized two-way crossover study. Each subject received atorvastatin 10 mg every morning for 2 weeks and atorvastatin 10 mg every morning with cimetidine 300 mg four times a day for 2 weeks, separated by a 4-week washout period. Steady-state pharmacokinetic parameters (based on an enzyme inhibition assay) and lipid responses were compared. Results: Pharmacokinetic parameters and lipid responses were similar following administration of atorvastatin alone and atorvastatin with cimetidine. Mean values for C_max (the maximum concentration) were 5.11 ng · eq · ml⁻¹ and 4.54 ng eq · ml⁻¹, for t_max (the time to reach maximum concentration) 2.2 h and 1.3 h, for AUC_0–24 (area under the concentration-time curve from time 0 h to 24 h) 58.6 ng eq · h · ml⁻¹ and 58.5 ng eq · h · ml⁻¹, and for t_1/2 (terminal half-life) 10.1 h and 17.0 h, respectively, following administration of atorvastatin alone and atorvastatin with cimetidine. Following treatment with atorvastatin alone and atorvastatin with cimetidine, mean values for the percentage change from baseline for total cholesterol were −29.5% and −29.9%, for low-density lipoprotein (LDL) cholesterol −41.0% and −42.6%, for high-density lipoprotein (HDL) cholesterol 6.3% and 5.8%, and for triglycerides −33.8% and −25.8%, respectively. Conclusions: The rate and extent of atorvastatin absorption and the effects of atorvastatin on LDL-cholesterol responses are not influenced by coadministration of cimetidine. Received: 17 February 1997 / Accepted in revised form: 3 November 1997     

Involvement of multiple cytochrome P450 isoforms in naproxen O-demethylation

Objective: A series of studies was undertaken to determine the cytochrome P450 isoform(s) involved in naproxen demethylation and whether this included the same isoforms reported to be involved in the metabolism of other NSAIDs. Methods: (S)-Naproxen was incubated with human liver microsomes in the presence of a NADPH-generating system and the formation of desmethylnaproxen was measured by high-performance liquid chromatography (HPLC). To further clarify the specific isoforms involved, experiments were conducted with preparations expressing only a single P450 isoform (vaccinia virus-expressed cells and microsomes derived from a lymphoblastoid cell line, each transfected with specific P450 cDNAs) as well as inhibition studies using human liver microsomes and putative specific P450 inhibitors. Results: In human liver microsomes (n=7), desmethylnaproxen formation was observed with a mean k_M of 92 (21) μmol · l⁻¹, V_max of 538 pmol · min⁻¹ · mg⁻¹ protein and C_int2 (reflective of a second binding site) of 0.36 μl · min⁻¹ · mg⁻¹ protein. This C_int2 term was added since Eadie-Scatchard analysis suggested the involvement of more than one enzyme. Studies using putative specific P450 inhibitors demonstrated inhibition of this␣reaction by sulfaphenazole, (apparent Ki= 1.6 μmol · l⁻¹), warfarin (apparent Ki=27 μmol · l⁻¹), piroxicam (apparent Ki=23 μmol · l⁻¹) and tolbutamide (apparent Ki=128 μmol · l⁻¹). No effect was observed when α-naphthoflavone and troleandomycin were employed as inhibitors, but reaction with furafylline produced, on average, a maximum inhibition of 23%. At a naproxen concentration of 150 μmol · l⁻¹, formation of desmethylnaproxen was observed in cells expressing P450 1A2, 2C8, 2C9 and its allelic variant 2C9R144C. To further characterize these reactions, saturation kinetics experiments were conducted for the P450s 1A2, 2C8 and 2C9. The k_M and V_max for P450 1A2 were 189.5 μmol · l⁻¹ and 7.3 pmol · min⁻¹ · pmol⁻¹ P450, respectively. Likewise, estimates of k_M and V_max for P450 2C9 were 340.5 μmol · l⁻¹ and 41.4 pmol · min⁻¹ · pmol⁻¹ P450, respectively. Reliable estimates of k_M and V_max could not be made for P450 2C8 due to the nonsaturable nature of the process over the concentration range studied. Conclusion: Multiple cytochrome P450 isoforms (P450 1A2, 2C8 and 2C9) appear to be involved in naproxen demethylation, although 2C9 appears to be the predominant form. Received: 16 September 1996 / Accepted in revised form: 20 December 1996     

Reduction of cisplatin toxicity in cultured renal tubular cells by the bioflavonoid quercetin

Quercetin (QC), a polyphenolic compound widely distributed in fruits and vegetables has recently gained interest due to its cisplatin (CP) sensitizing properties in cancer cells. It is currently unknown, whether quercetin also increases the susceptibility of the kidneys to cisplatin toxicity. We studied the effects of various bioflavonoids on CP toxicity in an in vitro model of cultured tubular epithelial cells (LLC-PK₁). Viability of LLC-PK₁ cells, as assessed by lactate dehydrogenase (LDH) release and MTT-test, was affected by CP (100–400 μM) in a time and dose dependent fashion. Pretreatment of cells with QC for 3 h significantly reduced the extent of cell damage. The protective activity of QC was concentration dependent, starting at 10–25 μM and reaching a plateau between 50 and 100 μM. Other bioflavonoids (catechin, silibinin, rutin) did not diminish cellular injury, even at higher concentrations (100–500 μM). Quercetin itself showed some intrinsic cytotoxicity at concentrations exceeding 75 μM. Our data indicate that quercetin reduces cisplatin toxicity in cultured tubular epithelial cells. The exact mechanism of protection is unclear, though scavenging of free oxygen radicals may play an important role. Received: 26 January 1998 / Accepted: 16 March 1998     

Pharmacokinetics and pharmacodynamics of acetazolamide in patients with transient intraocular pressure elevation

Objective: To characterize the pharmacokinetics and pharmacodynamics of acetazolamide in patients with transient intraocular pressure (IOP) elevation and to provide individual patients with the optimal dosage regimen for this drug. Methods: We studied 17 patients with transient IOP elevation, who were given 62.5–500 mg acetazolamide orally as single or repetitive doses. Plasma acetazolamide concentration and IOP were measured at approximately 1, 3, 5, and 9 h after the last acetazolamide administration. Pharmacokinetics and pharmacodynamics were analyzed by nonlinear mixed-effect modeling using the program NONMEM. Results: The plasma concentration profile of acetazolamide was characterized by a one-compartment model with first-order absorption. The apparent oral clearance was related to the creatine clearance (CCR) which was estimated by the Cockcroft and Gault equation, as follows: 0.0468 · CCR l · h⁻¹. The estimated apparent oral volume of distribution, first-order absorption rate constant, and absorption lag time were 0.231 l · kg⁻¹, 0.821 · h⁻¹, and 0.497 h, respectively. IOP after oral acetazolamide administration was characterized by an E_max model. The maximal effect in lowering the IOP (E_max) was 7.2 mmHg, and the concentration corresponding to 50% of the maximal effect (EC₅₀) was 1.64 μg · ml⁻¹. As 70% of E_max was achieved at a plasma concentration of 4 μg · ml⁻¹, this concentration was considered satisfactory for lowering IOP. The recommended dosage was calculated so that the minimum plasma concentration at steady state exceeded this target concentration; 250 mg t.i.d., 125 mg t.i.d., 125 mg b.i.d., and 125 mg once daily for the patients with CCR values of 70, 50, 30, and 10 ml · min⁻¹, respectively. Conclusion: Measuring plasma concentrations of acetazolamide and subsequent pharmacokinetic and pharmacodynamic analyses are useful for estimating its concentration-dependent effectiveness in lowering the IOP in individual patients. The dosage regimen presented in this study is expected to improve the benefits of acetazolamide pharmacotherapy in most elderly patients with transient rises in IOP following intraocular surgery. Received: 10 April 1997 / Accepted in revised form: 21 October 1997     

Olfactory toxicity of methyl iodide in the rat

 The monohalomethanes (methyl iodide, methyl bromide and methyl chloride) are widely used industrial methylating agents with pronounced acute and chronic toxicity in both experimental animals and man. Recently inhalation exposure of rats to methyl bromide has been shown to result in severe olfactory toxicity. This study examined the effects on the rat nasal cavity of inhalation of methyl iodide (100 ppm for 0.5 – 6 h), and demonstrated that methyl iodide is a more potent olfactory toxin than methyl bromide. Within the nasal cavity the olfactory epithelium was the principle target tissue, and it was only at high doses (600 ppm.h) that limited damage to transitional epithelium occurred. The squamous and respiratory epithelia were consistently unaffected. Within olfactory epithelium the sustentacular cells were the primary cellular target and damage to sensory cells appeared to be a secondary event. Methyl iodide induced olfactory damage was reversible, and 2 weeks after exposure almost complete repair had taken place. Received: 7 March 1995 / Accepted: 3 May 1995