The in vivo evaluation of the antitumour effect using xenografted mice is a fundamental step in the preclinical development process of oncology drugs. In this type of experiments tumour cells from immortalized human cell lines are inoculated in athymic mice; the animals are then randomized in groups treated either with a vehicle or an active treatment and the corresponding tumour growth curves are compared for obtaining information on the efficacy of tested compounds. In this respect, one of the most usual metrics is the tumour growth inhibition. Tumour growth inhibition is calculated as difference between tumour weight in treated and control animals expressed as percentage of tumour weight in control animals. However, this metric is dose- and schedule-dependent and may be strongly influenced by the time-point at which the measurements are performed. We recently developed a simple and effective pharmacokinetic-pharmacodynamic model linking the plasma concentrations of anticancer compounds to the effect on the tumour growth (Simeoni et al. 2004). The model successfully described the growth of total tumour mass obtained in mice given anticancer drugs, providing physiologically relevant parameters. In this communication two examples of its application to a discovery candidate and to a known anticancer drug are shown. The dependencies of tumour growth inhibition on the experimental conditions are also discussed using simulations. The experiments performed using A2780 human ovarian carcinoma cell lines were implanted sub cuteinto the left flank of nude mice (n=6–10/group). At palpable tumour, mice received placebo or the active drug (Drug A or vinblastine). Tumour dimensions were measured by caliper and tumour weights were calculated, assuming unit density, as D · d2/2, where D and d are the larger and the smaller dimensions, respectively. The pharmacokinetics were investigated in separate groups (n=3–5) of tumour-bearing mice. The drug concentrations in plasma were measured using liquid chromatography with tandem mass spectrometry. Compartmental pharmacokinetic models were fitted to the plasma concentration-time data using non-linear regression (Winnonlin program, v. 3.1, Pharsight). The pharmacodynamic model is summarized in the scheme reported in fig. 1. It is considered that, in control animals (unperturbed growth), the tumour mass grows exponentially up to a threshold weight, above which the growth becomes linear with time. The model parameters λ0 and λ1 provide estimates of the rates of growth in these two phases. In the treated animals the growth is perturbed by the anticancer treatment that makes some cells non-proliferating, eventually bringing them to death. In this case, the tumour growth rate is decreased proportionally to both plasma drug concentrations and weight of proliferating tumour cells, via a parameter k2, which is representative of the potency of the anticancer agent. Cells affected by drug action stop proliferating and pass through different stages, characterized by progressive degrees of damage and, eventually, they die. This can be described using a transit compartmental system, characterized by a first order rate constant k1. In the model, the total tumour weight is obtained as the sum of the weights of proliferating and damaged cells. The model was simultaneously fitted to the average tumour mass data obtained in control and treated animals, using non-linear regression (Winnonlin, v. 3.1, Pharsight). Scheme of the pharmacokinetic-pharmacodynamic (PK-PD) model. Unperturbed growth: w(t): total tumour mass, w0: initial tumor mass, λ0: rate constant of exponential growth, λ1: slope of the linear growth, ψ: shape factor. Perturbed growth: Z1(t): proliferating tumour mass, Z2(t), Z3(t), Z4(t): mass of tumour cells in the different stage of damage, K2 proportionality constant representing the potency of the agent, K1 first-order transfer rate constant. Candidate Drug A. In this example, the model was used prospectively to simulate the outcome of subsequent studies. In a preliminary experiment, mice were given Drug A intraperitoneally, bid×13 days at a dose level of 45 mg/kg (fig. 2). Based on the known pharmacokinetics of Drug A, the model was fitted to the data and the following pharmacodynamic parameters were calculated: w0=0.008 g, λ0=0.019 h−1, λ1=0.029 g · hr−1, K1=0.13 hr−1, K2=7.3 · 10–6 ml · hr−1 · ng−1. It was subsequently decided to test the candidate using intravenous dosing. For this purpose it was considered interesting to use the model for predicting the outcome of three possible regimens (t.i.d.×1 day, q.d.×11 days, b.i.d.×4 days). Preliminary pharmacokinetic data after intravenous dosing were used to simulate the expected plasma profiles at the different dosing regimens and the corresponding tumour growth curves were generated using the pharmacodynamic parameters previously estimated from the intraperitoneal experiment. Predictions and observations are shown in fig. 3. Despite some quantitative differences, essentially due to the different growth of controls, the qualitative agreement between predictions and observations at the three different schedules was excellent. It has also to be considered that many factors can influence the response in case of different administration routes (e.g., first-pass formation of active metabolites), so that this approach may help in interpreting the occurrence of such complexities. Observed and model-fitted tumour growth curves obtained in nude mice given intraperitoneally Drug A (45 mg/kg b.i.d.×13 days from Day 8). Left panel. Tumour growth curves predicted in nude mice given intravenously Drug A at 60 mg/kg dose level either t.i.d.×1 day, q.d.×11 days or b.i.d.×4 days from Day 9. Predictions were based on PD parameters obtained after the intraperitoneal administration. Right panel. Tumour growth curves observed in nude mice given Drug A intravenously at 60 mg/kg dose level either t.i.d.×1 day, q.d.×11 days or b.i.d.×4 days from Day 9. Vinblastine. Mice were given either the vehicle or vinblastine intravenous in single dose or q.4d×2 at the dose level of 3 mg/kg. The pharmacokinetic analysis was performed using an ancillary group of animals. The good agreement between the observed and model-fitted tumour growth data in the different experimental arms is shown in fig. 4. Observed and model-fitted tumour growth curves obtained in nude mice given either the vehicle or vinblastine intravenously (3 mg/kg given either as a single dose or q.4d×2 from Day 8). In the inset the fitting of the pharmacokinetic data is shown. Considerations on the experimental design. For evaluating the dependence of tumour growth inhibition metric from the experimental design, the outcome of different intravenous treatments of vinblastine was simulated: single doses of 1 or 6 mg/kg or 1 mg/kg given q.4d×2. For each treatment the average tumour growth curves were generated using the data of the previous experiment and the corresponding tumour growth inhibition were calculated and plotted (fig. 5). Maximal values of tumour growth inhibition were reached at different time points and biased estimations would have been obtained in case tumour growth inhibition was measured at a fixed time (e.g., as typically done for cytotoxics, 7 days after the end of treatment). Lower panel. Tumour growth curves predicted in nude mice given either the vehicle or vinblastine intravenously (1 mg/kg either as a single dose or q.4d×2 and 6 mg/kg single dose from Day 8). Upper panel. Tumour growth inhibition versus time curves derived using the predicted data of the lower panel. Further examples of the successful application of a pharmacokinetic-pharmacodynamic approach for modeling and predicting the tumour growth in xenografted mice have been presented. Compared to experimental tumour growth inhibition, the model is able to provide parameters that are not dependent on the experimental conditions. Thus they can be used prospectively for predicting the outcome of the subsequent experiments based on preliminary results. The model can be used in support to oncology research for ranking compounds and optimizing the experimental designs, allowing consistent savings in terms of resources and time. The authors would like to thank the scientists of the bioanalytical department involved in the generation of the pharmacokinetic data.

Polymeric cytotoxic conjugates are being developed with the aim of preferential delivery of the anticancer agent to tumour. MAG-CPT comprises the topoisomerase I inhibitor camptothecin linked to a water-soluble polymeric backbone methacryloylglycynamide (average molecular weight 18 kDa, 10% CPT by weight). It was administered as a 30-min infusion once every 4 weeks to patients with advanced solid malignancies. The objectives of our study were to determine the maximum tolerated dose, dose-limiting toxicities, and the plasma and urine pharmacokinetics of MAG-CPT, and to document responses to this treatment. The starting dose was 30 mg m −2 (dose expressed as mg equivalent camptothecin). In total, 23 patients received 47 courses at six dose levels, with a maximum dose of 240 mg m −2 . Dose-limiting toxicities were myelosuppression, neutropaenic sepsis, and diarrhoea. One patient died after cycle 1 MAG-CPT at the maximum dose. The maximum tolerated dose and dose recommended for further clinical study was 200 mg m −2 . The half-lives of both MAG-CPT and released CPT were prolonged (>6 days) and measurable levels of MAG-CPT were retrieved from plasma and urine 4 weeks after treatment. However, subsequent pharmacodynamic studies of this agent have led to its withdrawal from clinical development.

Polymeric drug conjugates are a new and experimental class of drug delivery systems with pharmacokinetic promises. The antineoplastic drug camptothecin was linked to a water-soluble polymeric backbone (MAG-CPT) and administrated as a 30 min infusion over 3 consecutive days every 4 weeks to patients with malignant solid tumours. The objectives of our study were to determine the maximal tolerated dose, the dose-limiting toxicities, and the plasma and urine pharmacokinetics of MAG-CPT, and to document anti-tumour activity. The starting dose was 17 mg m −2 day −1 . Sixteen patients received 39 courses at seven dose levels. Maximal tolerated dose was at 68 mg m −2 day −1 and dose-limiting toxicities consisted of cumulative bladder toxicity. MAG-CPT and free camptothecin were accumulated during days 1–3 and considerable amounts of MAG-CPT could still be retrieved in plasma and urine after 4–5 weeks. The half-lives of bound and free camptothecin were equal indicating that the kinetics of free camptothecin were release rate dependent. In summary, the pharmacokinetics of camptothecin were dramatically changed, showing controlled prolonged exposure of camptothecin. Haematological toxicity was relatively mild, but serious bladder toxicity was encountered which is typical for camptothecin and was found dose limiting.

Camptothecin (CPT) is a potent, antitumour drug acting mainly through inhibition of topoisomerase I during the S-phase of the cell cycle. Despite its impressive antitumour activity, clinical development was halted for unpredictable toxic events. Two soluble N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers were synthesised to contain CPT (5 wt.% and 10 wt.%). CPT was covalently linked at its alpha-hydroxyl group to the polymers through a Gly-Phe-Leu-Gly- spacer. In-vitro, CPT-conjugates were fairly resistant to hydrolysis in plasma as in buffer at neutral pH (0.2-0. 4% free CPT/h), while elastase and cysteine-proteases were able to release the active drug. Plasma levels in mice after intravenous administration of CPT-conjugates confirmed the modest hydrolysis in plasma. Plasma levels were approximately 5-fold lower than those observed at the highest tolerated dose of CPT administered in classical vehicles. Biodistribution in HT29 human colon carcinoma bearing mice was carried out after i.v. injection of [3H]CPT-conjugate and free [3H]CPT. Radioactivity uptake in tumour was evident only after [3H]CPT-conjugate treatment. Repeated intravenous administration of CPT-conjugates to HT29-bearing mice gave more than 90% tumour inhibition, some complete tumour regressions and no toxic deaths. The improved pharmacological profile on HT29 human colon carcinoma xenografts of the first poly(HPMA)-CPT conjugates might be ascribed to their prolonged intra-tumour retention and sustained release of the active drug.

OBJECTIVES To examine the pharmacokinetic characteristics of the selective norepinephrine reuptake inhibitor, reboxetine, in elderly patients with depression. PATIENTS Twelve female inpatients (mean age 80 +/- 4 years) with major depressive or dysthymic disorder were enrolled in a 4-week uncontrolled study of oral reboxetine 2-8 mg/day. METHODS After a one-week washout period, patients were randomized into two groups (groups A and B, n = 6/group). Reboxetine was given twice daily, starting with 2 mg/day during week 1 and increasing by 2 mg/day each week to 8 mg/day in week 4. Pharmacokinetic evaluations were carried out at two dosage levels in each group: at the end of weeks 1 and 3 in group A (2 and 6 mg/day), and at the end of weeks 2 and 4 in group B (4 and 8 mg/day). Blood and urine samples were taken for determination of reboxetine pharmacokinetics. RESULTS Reboxetine displayed linear pharmacokinetics, with dose-proportional changes, in elderly depressed patients. Mean total urinary recovery ranged from 4.06 to 6.17%. The mean area under the plasma concentration-time curve (AUCtau) and the maximum plasma drug concentration (Cmax) showed considerable variation between patients; at a dosage of 4 mg/day, AUCtau was 1,466-6,866 ngxh/ml and Cmax ranged from 169 to 663 ng/ml. CONCLUSIONS The pharmacokinetics of reboxetine are linear across the dosage range of 2-8 mg/day in elderly depressed patients, although Cmax and AUCtau values are higher (and more variable) than in young adults. These results support the use of a lower starting dose (4 mg/day) of reboxetine in the elderly.

Background: Reboxetine [(R,S)-2[(R,S)-alpha-(2-ethoxyphenoxy)benzyl]morpholine methanesulfonate] is a racemic compound that consists of equal proportions of R,R- and S,S-enantiomers. This study investigated the hemodynamic effects of reboxetine and the R,R-enantiomer compared with placebo in volunteers, The pharmacokinetics of reboxetine and its enantiomers were also investigated in the study.Methods: Nine healthy, male volunteers received single doses of 4 mg reboxetine, 2 mg R,R-enantiomer, and placebo at weekly intervals. Reboxetine and the R,R-enantiomer were well tolerated in all volunteers,Results: The heart rates of patients in the supine and standing positions were increased after reboxetine administration compared with the R,R-enantiomer (P < .05, except supine heart rate at 6 hours) and placebo (P < .05), Supine systolic and diastolic blood pressure was also increased by 3 +/- 4 and 1 +/- 4 mm Hg respectively, after reboxetine compared with R,R-enantiomer (-2 +/- 4 and 4 +/- 3 mm Hg) and placebo (-4 +/- 4 and -4 +/- 4 mm Hg) administration. The systolic and diastolic blood pressure measurements for subjects while standing did not differ significantly among treatments. There was no significant difference between the maximum plasma concentration, mean time to maximum plasma concentration, plasma half-life, or area under the plasma concentration-time curve (AUC) of the R,R-enantiomer after reboxetine or R,R enantiomer administration. The ratio of the mean AUC values for the R,R- and S,S-enantiomers was 2,1,Conclusion: These findings suggest that the S,S-enantiomer is responsible for the hemodynamic effects of reboxetine in humans. Increases in supine blood pressure after reboxetine administration may be interpreted as regression to the mean value and not caused by any treatment effect.

The absolute bioavailability of reboxetine enantiomers was assessed in six male and six female volunteers. In a two-way crossover study, subjects received 1.0 mg reboxetine orally and 0.3 mg reboxetine as an intravenous bolus. The R,R(-) and S,S(+) enantiomers in serial plasma and urine samples were determined by a validated LC-MS-MS method. There were no significant differences between treatments for clearance or dose-corrected AUC(0-infinity) values. The absolute bioavailability was 0.919 and 1.02 for R,R(-) reboxetine and S,S(+) reboxetine, respectively. A secondary objective of the study was to assess gender effects on pharmacokinetics of the enantiomers. Significant differences in volume of distribution between genders were observed, but differences in weight-corrected volumes were not significant. Weight-corrected systemic clearance and oral clearance tended to be lower in males, but this difference reached statistical significance only for weight-corrected oral clearance of R,R(-) reboxetine. C(max) after oral administration was 40 and 48% higher in women than men for R,R(-) reboxetine and S,S(+) reboxetine, respectively. These results indicate that reboxetine enantiomers are well absorbed after oral administration and that little first-pass metabolism occurs. There are no clinically significant effects of gender on the pharmacokinetics of reboxetine enantiomers.

The effect of repeated administration of rifabutin on the pharmacokinetics and metabolism of ethambutol was evaluated in ten healthy volunteers. The subjects received a single oral administration of 1200 mg ethambutol on days 1 and 10 and a single daily oral dose of 300 mg rifabutin from days 3 to 9. No statistically significant difference was found in plasma pharmacokinetics (C(max), t(max), AUC, half-life and MRT) and in the renal clearance, whereas a significant decrease in the amount of unchanged ethambutol excreted in urine was observed. The decrease observed in ethambutol urinary excretion may be accounted for by taking into consideration the variability of the urinary excretion of ethambutol reported in the literature. However, a slight, likely not clinically relevant, induction or activation of kidney alcohol and/or aldehyde dehydrogenase isoenzymes by rifabutin cannot be ruled out at present. Evidence exists in the present study for autoinduction of rifabutin metabolism; this is shown by the lower plasma concentrations obtained 24 h after the seventh dose as compared to the theoretical concentrations.

Reboxetine, (RS)-2-[(RS)-alpha-(2-ethoxyphenoxy)benzyl]morpholine methanesulphonate, is a racemic compound and consists of a mixture of the (R,R)- and (S,S)-enantiomers. The pharmacokinetics of reboxetine enantiomers were determined in a crossover study in three male beagle dogs. Each animal received the following oral treatments, separated by 1-week washout period: 10 mg/kg reboxetine, 5 mg/kg (R,R)- and 5 mg/kg (S,S)-. Plasma and urinary levels of the reboxetine enantiomers were monitored up to 48 h post-dosing using an enantiospecific HPLC method with fluorimetric detection (LOQ: 1.1 ng/ml in plasma and 5 ng/ml in urine for each enantiomer). After reboxetine administration mean tmax was about 1 h for both enantiomers. Cmax and AUC were about 1.5 times higher for the (R,R)- than for the (S,S)-enantiomer, mean values +/- SD being 704 +/- 330 and 427 +/- 175 ng/ml for Cmax and 2,876 +/- 1,354 and 1,998 +/- 848 ng.h/ml for AUC, respectively. No differences between the (R,R)- and (S,S)-enantiomers were observed in t1/2 (3.9 h). Total recovery of the two enantiomers in urine was similar, the Ae (0-48 h) being 1.3 +/- 0.7 and 1.1 +/- 0.7% of the enantiomer dose for the (R,R)- and the (S,S)-enantiomers, respectively. No marked differences in the main plasma pharmacokinetic parameters were found for either enantiomer on administration of the single enantiomers or reboxetine. No chiral inversion was observed after administration of the separate enantiomers, as already observed in humans.

The pharmacokinetics of reboxetine have been investigated in 12 healthy male volunteers after a single 2 mg dose of reboxetine and at steady state, following the last administration of a multiple-dose regimen (2mg twice a day for 5 1/2 d). Reboxetine was analysed in plasma and urine samples collected up to 72 h post-dosing using an HPLC method with UV detection. The urinary excretion rate of 6-beta-hydroxycortisol, used as a marker for cytochrome P450IIIA activity, was also tested, and any possible alteration was correlated to reboxetine plasma levels. The dosing regimen was well tolerated by all subjects. Reboxetine pharmacokinetic parameters, calculated after the single dose using non-compartmental analysis, were in good agreement with those obtained in previous studies. Following the multiple-dosing regimen, no significant deviations from expectation based on linear superposition was observed. The accumulation ratio, based on repeated-dose/single-dose ratios of Cmax, AUC(0-12 h), and C(12 h) was approximately two. A slight rise was recorded for the average excretion rate of 6-beta-hydroxycortisol over 48 h by the end of treatment; however, the difference was not statistically significant and the mean excretion rates were within the normal reference range. Thus a significant induction of P450IIIA was not indicated.

The pharmacokinetics of flosequinan and its active metabolite, flosequinoxan, were investigated following a single 100mg oral dose in 10 patients with compromised hepatic function. Plasma and urine samples were collected for up to 144 h postdose and analyzed by HPLC. All 10 patients provided analyzable data even though one patient withdrew before the 144-h sample because of an adverse event unrelated to the study medication. Interpatient variability was appreciable for the plasma and urine concentrations was well as for the calculated pharmacokinetic parameters. Relative to a comparative cohort of normal subjects, flosequinan concentrations in the study patients were elevated, showing increases in mean AUC0-< (62.8 ± 49.4 vs 3.4 ± 1.5 ng-h/mL), AUCo-» (70.2 ± 58.3 vs 3.8 ± 1.6 ng-h/mL), Cˆ (2.43 ± 0.56 vs 1.30 ± 0.39 /tg/mL), and tV2 (20.7 ± 16.8 vs 1.7 ± 0.5 h). The mean systemic clearance decreased (47.3 ± 46.5 vs 544 ± 279 mL/min), along with the elimination rate constant (0.066 ± 0.069 vs 0.44 ± 0.13 h-1). Mean flosequinoxan AUCo-/ and AUC0.o= values were unaffected by hepatic dysfunction. The mean time to peak was longer (36.4 ± 27.4 vs 7.0 ± 3.1 h) and cmax was less (0.98 ± 0.52 vs 1.84 ± 0.26 /tg/mL) than in normal subjects. These findings are consistent with a decrease in the rate of flosequinan metabolism to flosequinoxan. Five patients reported adverse events, which included headache (three patients) and syncope (one patient). Thus the consequences of hepatic dysfunction are a longer flosequinan elimination time, resulting in elevated flosequinan plasma levels, and a decrease in the rate flosequinoxan formation. This decrease in metabolic rate leads to an increased exposure to total quinolones (72 % more) for patients with hepatic dysfunction than for normal subjects. On the basis of the results of this study, it is suggested that dosage adjustment should be considered in patients with hepatic dysfunction.

In this study, an open, double-blind, randomized, two-period, two-group crossover design was conducted in 14 healthy volunteers to study the bioequivalence of a fixed-dose generic product. After administration of test or reference products to each volunteer, both active ingredients were determined simultaneously in plasma samples using a developed and validated HPLC-UV method, and pharmacokinetic parameters, including Cmax, Tmax, AUC0–t, AUC0–∞, terminal elimination rate constant (λz), volume of distribution in steady state (Vd(ss)), mean residence time (MRT), clearance (Cl), terminal elimination rate constant (Kel) were determined in each subject using the standard non-compartmental approach. Statistical comparison showed that the test and reference products were bioequivalent in terms of both the rate and extent of bioavailability of both active ingredients. Finally, a new parameter named range overlap index (ROI) was introduced for the first time in this study in order to judge about the overall bioequivalence of the combination products. This parameter indicates the extent in which the two CI90% ranges of each parameter for two active ingredients overlap with each other. The ROI is suggested to be equal or more than 50% for two combination products in order to be known as bioequivalent. The ROI values of the bioequivalence-indicating parameters were 61.90%, 84.6%, and 76.0% for Cmax, AUC0→12, and AUC0→∞, respectively, which are indicative for bioequivalence in all the cases.

The pharmacokinetics of reboxetine, a new antidepressant agent, were found to be close to linear in a crossover study comparing administration of single 2, 3, 4 and 5 mg capsule doses in 15 healthy male volunteers, and in the same study the capsules were bioequivalent to the proposed therapeutic tablet formulation (4mg). Kinetic analysis was based on HPLC assay of reboxetine in plasma and urine collected up to 72 h after each administration. Plasma levels indicated a rapid absorption ( t max ⋍2h) and an elimination half‐life of about 13 h. Clearance and volume of distribution were modest (ratios to bioavailability: CL/ F ⋍29 mL min −1 ; V z /F ⋍32L); urinary excretion was ∼9% of dose, corresponding to a renal clearance of only 3 mL min −1 (a value consistent with the rate of glomerular filtration of unbound drug). In vitro , binding to plasma proteins, estimated from radioactivity levels following dialysis of 14 C‐labelled reboxetine, appeared to be dominated by α 1 ‐acid glycoprotein without marked saturation up to plasma concentrations of over 500 ng mL −1 (2.8–3.1% unbound with human plasma from three additional volunteers; 1.8–2.0% for 2gL −1 orosomucoid α 1 ‐acid glycoprotein, and 46.4–47.4% for 40 gL −1 albumin), whilst the mean C max in the current study was much lower (164 ng mL −1 after a 5 mg dose).

Reboxetine, (RS)-2-[(RS)-alpha-(2-ethoxyphenoxy)benzyl]morpholine methanesulphonate, is a racemic compound and consists of a mixture of the (R,R)- and (S,S)-enantiomers. In this study, brain and plasma levels of both enantiomers were determined in mice and rats after oral administration of reboxetine at doses (1.1 mg/kg, mouse; 20 mg/kg, rat) twice the respective ED50 values in the antireserpine test. Plasma and brain concentrations of each enantiomer were measured up to 6 h postdosing using an HPLC method with fluorimetric detection after derivatization with a chiral agent (FLEC). In mice and rats, brain and plasma levels of the (R,R)-enantiomer were always higher than those of the (S,S)-enantiomer. After normalization for dose, the mean AUC0-tz values of both the (R,R)- and (S,S)-enantiomers in mouse brain were about 23 and 32 times higher than in rat brain, respectively. In plasma, the corrected mean AUC0-tz values were about 5 (R,R) and 10 (S,S) times higher in mice than in rats. These results provide evidence for the higher bioavailability and/or lower clearance of both enantiomers in mice than in rats, and for a higher penetration of both enantiomers into mouse brain compared to rat brain.