Inhibition of paclitaxel elimination in the isolated perfused rat liver by Cremophor EL

Purpose: Cremophor can alter the pharmacokinetics of cytotoxic drugs, including doxorubicin and etoposide. In view of its presence in the formulation of paclitaxel, the aim of this study was to investigate the influence of Cremophor on the hepatobiliary elimination of paclitaxel. Methods: In a recirculating isolated perfused rat-liver system the elimination of 1.7 mg paclitaxel given as a bolus into the perfusate reservoir was monitored in perfusate and bile in controls and after the administration of either 80 or 800 μl Cremophor. The higher dose of Cremophor yields clinically relevant perfusate concentrations. Paclitaxel was measured in perfusate, bile, and liver tissue by high-performance liquid chromatography. Results: Cremophor caused a dose-dependent inhibition of the elimination of paclitaxel, with a statistically significant mean value ± SD, n = 3; (P < 0.05 versus controls Bonferroni t-test) 9-fold increase in AUC (2227±106 versus 245 ± 40 g ml⁻¹min), 9-fold decrease in total clearance (0.8±0.1 versus 7.0±1.1 ml/min), and 5-fold increase in elimination half-life (92±14 versus 18±4 min) being observed after a dose of 800 μl Cremophor. With the addition of Cremophor the amount of paclitaxel remaining after 3 h increased in perfusate from none to 20, increased in liver tissue from 4 to 18, and remained constant in bile at 11–13%. In the control group, 86 of the paclitaxel dose was recovered in bile as five putative metabolites, which were measured in paclitaxel equivalents, with the major metabolite. M3 co-eluting with 3′-p-hydroxypaclitaxel. This decreased to 45 of the dose on the addition of Cremophor, and the ratio of M3 to paclitaxel in bile decreased. Conclusions: Cremophor inhibits the hepatic elimination of paclitaxel in the isolated perfused rat liver, primarily by preventing the drug from reaching sites of metabolism and excretion. The presence of Cremophor in the paclitaxel formulation may therefore contribute to the nonlinear pharmacokinetics and pharmacodynamics of paclitaxel. Received: 24 February 1998 / Accepted: 22 June 1998     

Metabolism and biliary excretion of the novel anticancer agent 10-hydroxycamptothecin in the isolated perfused rat liver.

10-hydroxycamptothecin (HCPT), a natural analog of the alkaloid camptothecin (CPT), is a promising anticancer agent currently undergoing preclinical trials. Though HCPT is less toxic and more active in various human cancer cell lines and in animal tumor models than the clinically approved CPT-analog topotecan, little is known about its biotransformation products and their route of elimination. To investigate the metabolism and biliary excretion, livers of male Wistar rats were perfused with HCPT (5 microM). Bile and perfusate samples were collected for 60 min and quantified by reversed-phase high-performance liquid chromatography (HPLC). Besides HCPT, three metabolites, namely HCPT glucuronide (M1), hydroxyHCPT glucuronide (M2), and hydroxyHCPT (M3) could be identified by enzymatic hydrolysis with beta-glucuronidase and mass spectroscopy. Biliary secretion of HCPT and M1-M3 reached a peak secretion of 1532+/-124, 75+/-16, 5.8+/-1.6 and 2.1+/-0.5 pmoles/g liver.min, respectively, after 25 min. The total amount of HCPT and M1-M3 excreted into bile during the time of perfusion (60 min) was low and represented a mean of 9.9+/-3.2%, 0.44+/-0.17%, 0.041+/-0.010%, and 0.022+/-0.004% of the initial HCPT dose, respectively. In the perfusate, besides HCPT M1 and M2 but not M3 could be detected (maximum concentrations after about 20 min: 3248+/-210, 16.8+/-2.8 and 1.0+/-0.4 pmoles/g liver.min, respectively). The cumulative efflux of HCPT and M1 and M2 into the perfusate was 21.1+/-3.9%, 0.145+/-0.036% and 0.018+/-0.004% of the initial dose, respectively, indicating a preferable non-biliary secretion for HCPT and a predominant biliary elimination for conjugated HCPT biotransformation products. In conclusion, HCPT is biotransformed in a rat liver model to three metabolites, mainly excreted into bile, which may be of clinical relevance during cancer therapy.     

Metabolism of N -[2-(dimethylamino)ethyl]acridine-4-carboxamide in cancer patients undergoing a phase I clinical trial

N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (DACA) is an experimental antitumour agent that has just completed phase I clinical trials in New Zealand and the United Kingdom. Urine (0–72 h) was analysed from 20 patients receiving DACA infused over 3 h (dose range 60–1000 mg/m², the latter being the highest dose achieved in the trial). Aliquots were analysed for DACA and its metabolites by high-performance liquid chromatography (HPLC). Over 72 h, 44 ± 5% (range 20–60%) of the dose was recovered in the urine, with 0.8 ± 0.3% (range 0–3.1%) occurring as DACA. The major urinary metabolite was DACA-N-oxide-9(10H)acridone, accounting for 34 ± 3% of the dose. Minor metabolites were identified as N-mono- methyl-DACA-9(10H)acridone (2.0 ± 0.5%), DACA-9(10H)acridone (3.3 ± 0.5%), N-monomethyl-DACA (0.2 ± 0.1%) and DACA-N-oxide (0.5 ± 0.1%). No ring-hydroxylated metabolite was detected. The urinary excretion of metabolites was greatest over 0–6 h in most patients. The composition of urinary metabolites was also independent of the delivered dose. Plasma was sampled at intervals throughout the infusion and at time points up to 48 h post-administration. The major plasma metabolites observed were DACA-9(10H)acridone and DACA-N-oxide-9(10H)acridone. These results indicate that, based on urinary excreted metabolites, the major biotransformation reactions for DACA in humans involve N-oxidation of the tertiary amine side chain and acridone formation, both of which appear to be detoxication reactions. Received: 21 August 1998 / Accepted: 10 December 1998     

Hyperthermic intraperitoneal doxorubicin: pharmacokinetics, metabolism, and tissue distribution in a rat model

Background: The cytotoxic effect of several anticancer agents, including doxorubicin, can be enhanced by hyperthermia. The purpose of this study was to evaluate the effect of hyperthermia on the pharmacokinetics, metabolism, and tissue distribution of intraperitoneal (i.p.) doxorubicin in a rodent model. Methods: Doxorubicin was given i.p. to 20 Sprague-Dawley rats at a dose of 2 mg/kg over 60 min. Rats were randomized into two groups according to the temperature of the peritoneal perfusate: group NT received normothermic (37 °C) i.p. doxorubicin; group HT received hyperthermic (43 °C) i.p. doxorubicin. During the course of i.p. chemotherapy, peritoneal fluid and blood were sampled every 10 min. At the end of the procedure, rats were sacrificed and tissue samples (liver, spleen, small bowel, omentum, bladder, diaphragm, abdominal wall, heart) were collected. Concentrations of doxorubicin and its aglycone metabolites were determined in peritoneal fluid, plasma, and tissues by HPLC. Results: No significant differences in areas under the curve (AUC) of peritoneal fluid doxorubicin and plasma doxorubicin were found between group NT and group HT. AUC ratios (AUC peritoneal fluid/AUC blood) were 87.9 for group NT and 82.9 for group HT. Group HT exhibited increased doxorubicin concentrations for all intraabdominal tissues. These differences were significant for spleen (P = 0.03), small bowel (P = 0.03), and omentum (P = 0.03). Doxorubicin aglycone was detected in plasma of both groups within the first 10 min of the procedure. There was a significant (P < 0.001) increase in plasma aglycone AUC for group HT when compared with group NT. Group HT exhibited increased aglycone concentration for all tissues. This difference was significant for liver (P < 0.001) and bladder (P < 0.001). Conclusion: Hyperthermia did not affect significantly the pharmacokinetics of i.p. doxorubicin. Tissue concentrations of doxorubicin in small bowel, omentum, and spleen were significantly increased when the drug was administered by hyperthermic i.p. perfusion. Hyperthermia increased significantly the doxorubicin aglycone concentrations in plasma, liver, and bladder. Received: 14 August 1996 / Accepted: 12 May 1997     

Antitumor and antivascular effects of AC-7700, a combretastatin A-4 derivative,against rat liver cancer

Background. Unlike the many chemotherapeutic agents that do not effectively stop blood flow or induce necrosis in hepatocellular carcinoma, AC-7700 has been shown to inhibit tubulin polymerization and selectively stop tumor blood flow. The aim of this study was to elucidate the antivascular and antitumor effects of AC-7700 on rat hepatoma. Methods. AH-130 cells, a rat hepatoma cell line, were solidified and implanted into the liver of Donryu rats. Vascularity of the liver tumor was directly identified by in-vivo fluorescence microscopy from 0 to 60 min after the injection of 10 mg/kg AC-7700. To observe the antivascular effect of AC-7700, the vascular density of the tumor was measured and assessed as the ratio of preinjection to postinjection values. The antitumor effects were evaluated with histopathologic findings and analysis of animal survival. Results. In-vivo microscopic observation showed that tumor perfusion diminished within 30 min after AC-7700 administration. Vascular density in the AC-7700 group was significantly less than that in the control group at 60 min (AC-7700, 26.3 ± 16.4%; control, 88.5 ± 9.2%; P < 0.001). After AC-7700 injection, marked necrosis of tumor cells was observed histologically, and tumor area was decreased significantly (AC-7700, 11.5 ± 15.4 mm²; control, 43.5 ± 18.3 mm²; P < 0.05). The survival rate (50%) of the AC-7700 group animals was better than that of the control group (0%; P < 0.01). Conclusion. Markedly decreased tumor perfusion was induced by AC-7700 within 30 min, and this decrease may have contributed to the tumor necrosis and favorable outcome in the treatment group. AC-7700 appears to be a promising agent for the treatment of hepatocellular carcinoma. Received: September 14, 2001 / Accepted: February 21, 2002     

Comparative brain tissue distribution of camptothecin and topotecan in the rat

Purpose: The primary objective of this investigation was to compare the extent of brain distribution of the lactone and the carboxylate forms of camptothecin (CPT) and topotecan (TPT) in awake freely moving rats. Methods: The plasma concentration-time profiles of the lactone and the carboxylate forms of CPT and TPT were determined simultaneously after a single i.v. administration of the lactone form of each drug. Also, the brain extracellular fluid (ECF) concentration-time profiles were characterized utilizing the microdialysis technique. This technique allowed serial sampling of the brain ECF in awake rats. Results: CPT-lactone in plasma declined biexponentially with a terminal half-life of 102 ± 25.2 min. During the elimination phase, the plasma concentration of CPT-carboxylate was approximately ten times the concentration of CPT-lactone. The brain ECF to plasma distribution ratio measured as the ratio of the AUC in the brain ECF to the AUC in plasma was 0.51 ± 0.08 for CPT-lactone, and 0.26 ± 0.21 for CPT-carboxylate. The terminal half-life for TPT-lactone was 64.0 ± 9.4 min. During the elimination phase, the TPT-carboxylate concentration was higher than that of TPT-lactone but the carboxylate to lactone concentration ratio was much lower than that of CPT. The brain ECF to plasma distribution ratio was 0.38 ± 0.12 for TPT-lactone, and 0.21 ± 0.06 for TPT-carboxylate. Conclusions: CPT and TPT are distributed to the brain ECF most probably by passive diffusion across the blood-brain barrier. Although the brain ECF to plasma distribution ratio for CPT-lactone was higher than that for TPT-lactone, the brain ECF concentrations of TPT-lactone were significantly higher than the CPT-lactone brain ECF concentrations. The relatively high brain ECF to plasma distribution ratio of these two drugs makes them potential candidates for first-line treatment of CNS tumors. Received: 4 May 1998 / Accepted: 5 August 1998     

Effect of filgrastim on the pharmacokinetics of MX2 hydrochloride in patients with advanced malignant disease

Purpose: To investigate the effect of granulocyte colony-stimulating factor (G-CSF) on the pharmacokinetics and pharmacodynamics of the new morpholino anthracycline drug MX2. Methods: A total of 25 patients with advanced malignant disease participated in a dose-escalation study in the first cycle of treatment given i.v. at doses of 50–80 mg/m² (74–152 mg) with concomitant filgrastim (G-CSF, 5 μg/kg) given daily beginning at 24 h after the dose of MX2. Results: The mean fast distribution half-life (1.5 ± 1.0 min) and the mean plasma clearance (2.18 ± 0.95 l/min) were significantly lower than the respective mean values found in a previous study in which 27 patients had received MX2 (16.8–107.5 mg) alone (3.3 ± 2.2 min and 2.98 ± 1.68 l/min, respectively; P < 0.05). There was no correlation between plasma clearance and the delivered dose for the combined MX2-alone and MX2-filgrastim groups, indicating that the lower clearance observed in the G-CSF group was probably not due to the higher dose. Elimination half-lives of the metabolites M1 and M4 were significantly greater in the filgrastim group (19.8 ± 14.7 and 11.8 ± 5.0 h for M1 and 14.8 ± 4.1 and 12.3 ± 6.3 h for M2, respectively). Unlike the MX2-alone group, there was no relationship in the MX2- filgrastim group between the relative nadir neutrophil count and the dose or between the duration of grade IV neutropenia and the dose of MX2. Conclusions: This study shows that filgrastim decreased the plasma clearance of MX2 by approximately 25%, possibly by inhibition of metabolism. Received: 1 June 1997 / Accepted: 17 September 1997     

Metabolism of UCN-01 in isolated perfused rat liver: role of Mrp2 in the biliary excretion of glucuronides

UCN-01 is a promising, novel kinase inhibitor currently undergoing clinical development. Though UCN-01 shows pronounced antitumor activity, its metabolism and hepatic transport system is still unknown. To investigate the biotransformation and biliary excretion of UCN-01, livers of Wistar and Mrp2-deficient TR- rats were perfused with UCN-01 (0.2 microM) in a single pass system. In bile and perfusate, native UCN-01 and 5 novel metabolites (M1-M5) were quantified by HPLC and identified as glucuronides by enzymatic hydrolysis with beta-glucuronidase and mass spectroscopy. Cumulative efflux of UCN-01 and its metabolites M1-M5 into perfusate of Wistar rats was low (<0.14%) whereas total biliary excretion was up to 53-fold higher, representing 1.74, 0.54, 0.21, 1.17, 0.85 and 0.52% of infused UCN-01, respectively. After 60 min of perfusion, liver cells still contained approximately 95% of applied UCN-01. Biliary excretion greatly differs in TR- rats. While cumulative biliary excretion of UCN-01 and its metabolites M1-M5 was significantly reduced to 8.3, 5.3, 31.8, 10.4, 13.2 and 7.8%, efflux into perfusate was increased up to 2.2-fold. This indicates that in control rats, UCN-01 and its glucuronides are almost exclusively eliminated into bile by Mrp2. In summary, UCN-01 is extensively metabolized in the rat liver to 5 novel glucuronides mainly excreted into bile by Mrp2. Metabolism and biliary excretion of UCN-01 must be taken into consideration also during cancer therapy of patients.     

Inhibition of etoposide elimination in the isolated perfused rat liver by Cremophor EL and Tween 80

 Cremophor EL, a surfactant used in the clinical formulation of cyclosporine and paclitaxel, will reverse the multidrug resistance (MDR) phenotype in vitro. As other MDR modulators can alter the pharmacokinetics of cytotoxic drugs, the aim of this study was to examine the effect of Cremophor and another MDR-reversing surfactant, Tween 80, on the hepatic elimination and biliary excretion of etoposide. Using the isolated perfused rat-liver model with 80 ml recirculating perfusate containing 20% red blood cells and 4% bovine serum albumin, etoposide (1.6 mg) with and without Cremophor (800 or 80 mg) or Tween 80 (80 mg) was given into the perfusate reservoir, and perfusate and bile samples were collected for 3 h. Etoposide was measured by high-performance liquid chromatography (HPLC) and Cremophor was measured using a bioassay. Both surfactants changed the etoposide elimination profile from biphasic to monophasic. High-dose Cremophor increased the AUC (from 334±23 to 1540±490 μg min ml^-1, P<0.05) and decreased the total clearance (from 4.8±0.3 to 1.1±0.3 ml/min, P<0.05) and biliary clearance (from 2.6±1.1 to 0.5±0.2 ml/min, P<0.05) but decreased the elimination half-life (from 62±17 to 40±5 min, P<0.05) and volume of distribution (from 424±85 to 65±19 ml, P<0.05). Low-dose Cremophor and Tween 80 caused intermediate effects on these parameters that were statistically significant for total clearance, half-life, and volume of distribution. Cremophor had no adverse effect on liver function, whereas Tween 80 caused haemolysis and cholestasis. The initial high-dose Cremophor perfusate concentration was 0.8 mg/ml, which previous studies have shown to be clinically relevant and close to the optimal level for MDR reversal in vitro (1.0 mg/ml). Cremophor may be a clinically useful MDR modulator, but it may alter the pharmacokinetics of the cytotoxic drug. Received: 5 January 1995/Accepted: 25 August 1995     

Pharmacokinetics of intraperitoneal hyperthermic perfusion with mitoxantrone in ovarian cancer

Purpose: Theoretical data and experimental assumptions indicate that intraperitoneal hyperthermic chemotherapy may play a role in the treatment of peritoneal carcinomatosis. The feasibility, tolerability and pharmacokinetics of intraperitoneal hyperthermic perfusion with mitoxantrone were studied in patients with pretreated ovarian cancer. Methods: After cytoreductive surgery, 11 patients underwent intraperitoneal hyperthermic perfusion with mitoxantrone. A heated (42–43 °C) solution of the drug (28 mg/m²) was recycled through a perfusion apparatus into the abdominal cavity for 90 min. Treatment was repeated every month for two to four cycles. In six patients blood and peritoneal perfusate samples were collected at 0.5, 1, 1.5, 2, 4, 8, 16 and 24 h after drug administration and mitoxantrone was assayed by an HPLC method. Results: Although treatment was generally well tolerated, all patients developed transient intestinal subocclusion. Maximal mitoxantrone plasma concentrations (C_max), times to C_max (T_peak) and area under the curves (AUC) were highly variable between subjects (C_max 14–337 ng/ml; T_peak 0.5–8 h; AUC 222–4130 ng · ml⁻¹ · h). The plasma to peritoneal fluid AUC ratio was significantly higher during the second (0.177) than during the first cycle (0.066), suggesting a cycle-dependent increase in systemic bioavailability. Furthermore, when comparing present data with those reported previously, hyperthermic perfusion may have lowered the mitoxantrone levels in the peritoneal fluid without greatly influencing plasma levels. Conclusions: Intraperitoneal mitoxantrone administered under hyperthermia to advanced ovarian cancer patients is feasible and well tolerated. Mitoxantrone pharmacokinetics may be altered by repeated intraperitoneal administration (increased bioavailability) and by hyperthermic perfusion (possibly, increased peritoneal tissue uptake). Received: 6 September 1999 / Accepted: 11 January 2000     

A pilot phase I trial of continuous hyperthermic peritoneal perfusion with high-dose carboplatin as primary treatment of patients with small-volume residual ovarian cancer

Purpose: Because intraperitoneal (i.p.) therapy may provide a therapeutic advantage and because hyperthermia enhances carboplatin (CBDCA) cytotoxicity, we evaluated the feasibility, toxicity, and pharmacokinetics of CBDCA given via continuous hyperthermic peritoneal perfusion (CHPP) in patients with small-volume residual ovarian cancer. Patients and Methods: Six patients underwent optimal cytoreductive procedures (residual disease ≤5 mm) as initial treatment of stages II and III epithelial ovarian adenocarcinoma. All patients received a 90-min CHPP at a CBDCA dose of 800–1200 mg/m², with the perfusate being recirculated rapidly from a reservoir through a heat exchanger, resulting in i.p. temperatures of 41–43 °C. Plasma, perfusate, and urine samples were collected and platinum was quantified by flameless atomic absorption spectrophotometry. Results: At no time did any patient's core temperature exceed 40 °C. Peak perfusate platinum concentrations were 8- to 15-fold higher than peak ultrafilterable plasma concentrations. The permeability-area product was extremely high and variable (14–90 ml/min), resulting in a regional advantage of 1.9–5.3. The percentage of the dose absorbed ranged widely from 27% to 77%. Dose-limiting hematologic toxicity was observed at a dose of 1200 mg/m² and this was associated with a CBDCA AUC in plasma of 11 mg min ml⁻¹. Conclusions: CHPP with CBDCA was safely given to three patients at a dose of 800 mg/m², and dose-limiting hematologic toxicities observed at 1200 mg/m², correlated with the plasma CBDCA exposure established when lower doses of CBDCA are given systemically. The pharmacokinetic data are consistent with the expected effect of vigorous mixing on the exposed peritoneal surface area. Variable drug absorption and clearance make the prediction of systemic exposure highly uncertain. These findings may have important implications for novel therapies given i.p. Received: 9 March 1998 / Accepted: 11 June 1998     

Effects of amifostine on perfused isolated rat heart and on acute doxorubicin-induced cardiotoxicity

Purpose: To determine the effects of amifostine on an isolated perfused rat-heart model and its protective activity with regard to cardiotoxic doxorubicin perfusion. Methods: Langendorff constant-pressure isolated rat-heart preparations were used to analyze the effects of the drugs during a 40-min period of perfusion after a 20-min stabilization interval. The first study was conducted with amifostine alone (controls and 10⁻⁶, 10⁻⁵, and 10⁻⁴ M amifostine; n=6 in each group). The second study was conducted with amifostine and doxorubicin (controls, 2.5 × 10⁻⁵ M doxorubicin, 2.5 × 10⁻⁵ M doxorubicin and 10⁻⁵ M amifostine, and 2.5 × 10⁻⁵ M doxorubicin and 10⁻⁴ M amifostine; n=4 in each group). Results: Amifostine had no significant effect on hemodynamic parameters at 10⁻⁶, 10⁻⁵, and 10⁻⁴ M concentrations. However, amifostine increased the coronary flow expressed as a percentage ± SEM of the baseline flow as follows: 82 ± 4% for controls, 95 ± 6% for 10⁻⁶ M amifostine, (P=0.13), 111 ± 4% for 10⁻⁵ M amifostine (P < 0.01), and 104 ± 3% for 10⁻⁶ M amifostine (P < 0.01). When we commenced an amifostine perfusion 20 min in advance of and then during a 40-min perfusion with doxorubicin, at a cardiotoxic concentration of 2.5 × 10⁻⁵ M the left ventricular pressures (LVDP, expressed as percentages ± SEM of the baseline LVDP before doxorubicin) were 55 ± 3% for the doxorubicin controls, 68 ± 2% for doxorubicin with 10⁻⁵ M amifostine (P=0.05), and 80 ± 3% for doxorubicin with 10⁻⁴ M amifostine (P < 0.01). Whether this protective effect might be related to the known free-radical-scavenging activity of amifostine remains to be determined. Conclusion: On a Langendorff-type model of rat heart, 10⁻⁵ and 10⁻⁴ M amifostine alone induced a coronary dilation and, when associated with a cardiotoxic concentration of 2.5 × 10⁻⁵ M doxorubicin, 10⁻⁵ and 10⁻⁴ M amifostine displayed a cardioprotective effect. Received: 9 March 1998 / Accepted: 6 July 1998     

Pharmacokinetics and pharmacodynamics of MX2 hydrochloride in patients with advanced malignant disease

The purpose of the present study was to investigate the pharmacokinetics and pharmacodynamics of the new morpholino anthracycline drug MX2. A total of 27 patients with advanced cancer participated in a dose-escalation study in the first cycle of treatment with drug given i.v. at doses of 10–50 mg/m² (total dose 16.8–107.5 mg). The mean total systemic plasma clearance (CL) of MX2 was 2.98 ± 1.68 l/min, the mean volume of distribution at steady state was 1460 ± 749 l and mean elimination half-life was 10.8 ± 5.1 h. The area under the plasma concentration-time curve (AUC) of MX2 was linearly related to the dose per kilogram and the dose per body surface area (r ² = 0.43, P < 0.01 and r ² = 0.44, P < 0.01, respectively). CL did not correlate with total body weight, lean body mass or body surface area. The mean elimination half-lives of the metabolites M1, M2, M3 and M4 were 11.8 ± 5.0, 21.9 ± 11.8, 19.0 ± 11.3 and 12.3 ± 6.3 h, respectively. The fractional E _max model produced a much better fit to the relative nadir neutrophil count versus dose data (r ² = 0.42) than to the relative nadir neutrophil count versus AUC or peak concentration (C _max) data (r ² = 0.15 and 0.09, respectively). There seemed to be a threshold dose of about 65 mg of MX2 at or above which a large proportion of patients had a nadir neutrophil count of less than 0.5 × 10⁹/l. This study shows that the pharmacokinetics of MX2 are similar to those of other anthracyclines. With other anthracyclines the degree of myelosuppression seems to depend more on the AUC and C _max than on the delivered dose; however, with MX2 the degree of myelosuppression depends more on the dose given than on drug exposure expressed as the AUC or C _max. Received: 18 February 1996 / Accepted: 20 December 1996     

Hepatic arterial infusion chemotherapy for unresectable confined liver metastases: prediction of systemic toxicity with the application of a scintigraphic and pharmacokinetic approach

Purpose: The incorrect positioning of the arterial Port-a-Cath or the presence of anatomic or functional hepatic arteriovenous shunting may explain the occurrence of systemic toxicity of hepatic arterial infusion of floxuridine in patients with liver metastases. The aim of our study was to predict the occurrence of systemic toxic effects from this treatment using a scintigraphic and pharmacokinetic approach. Methods: A group of 26 patients were studied. Before treatment, Tc-99m-labelled macroaggregated albumin arterial perfusion scintigraphy was performed to verify the correct positioning of the catheter, to evaluate the percentage of pulmonary uptake of the tracer, reflecting intrahepatic arteriovenous anatomic shunting, and to qualitatively assess the perfusion pattern of the metastases with respect to the normal liver parenchyma (SPECT images). Hepatic arteriovenous functional shunting was assessed through the bioavailability of intraarterially administered D-sorbitol. Treatment was then started and systemic toxic effects were evaluated according to WHO recommendations. Results: No correlation was found between anatomic shunting (≤10% in all patients) and systemic toxicity of treatment. The 9 patients with hypoperfused metastases experienced a significantly lower level of toxic effects (1 low-grade toxicity and 8 no toxicity) than the 17 with hyperperfused metastases (6 high-grade toxicity, 5 low-grade and 6 no toxicity; χ² = 7.170, P = 0.028). Functional shunting was significantly different in patients with high-grade, low-grade and no toxicity (46.5 ± 19.9%, 15.8 ± 12.7% and 16.5 ± 10.3%, respectively; P<0.001 by analysis of variance). Moreover, functional shunting was significantly greater only in patients with hyperperfused metastases who developed high-grade toxicity. Conclusions: A protocol combining scintigraphic and pharmacokinetic methods is of value in the individual patient in assessing the risk of high-grade systemic toxicity during hepatic arterial infusion of floxuridine. A flow-chart used in our ongoing prospective study for the evaluation of patients undergoing regional chemotherapy for liver metastases is included. Received: 3 December 1998 / Accepted: 28 April 1999     

Phase I and pharmacokinetic study of d-limonene in patients with advanced cancer

Purpose: d -Limonene is a natural monoterpene with pronounced chemotherapeutic activity and minimal toxicity in preclinical studies. A phase I clinical trial to assess toxicity, the maximum tolerated dose (MTD) and pharmacokinetics in patients with advanced cancer was followed by a limited phase II evaluation in breast cancer. Methods: A group of 32 patients with refractory solid tumors completed 99 courses of d-limonene 0.5 to 12 g/m² per day administered orally in 21-day cycles. Pharmacokinetics were analyzed by liquid chromatography-mass spectrometry. Ten additional breast cancer patients received 15 cycles of d-limonene at 8 g/m² per day. Intratumoral monoterpene levels were measured in two patients. Results: The MTD was 8 g/m² per day; nausea, vomiting and diarrhea were dose limiting. One partial response in a breast cancer patient on 8 g/m² per day was maintained for 11 months; three patients with colorectal carcinoma had prolonged stable disease. There were no responses in the phase II study. Peak plasma concentration (C_max) for d-limonene ranged from 10.8 ± 6.7 to 20.5 ± 11.2 μM. Predominant circulating metabolites were perillic acid (C_max 20.7 ± 13.2 to 71 ± 29.3 μM ), dihydroperillic acid (C_max 16.6 ± 7.9 to 28.1 ± 3.1 μM ), limonene-1,2-diol (C_max 10.1 ± 8 to 20.7 ± 8.6 μM ), uroterpenol (C_max 14.3 ± 1.5 to 45.1 ± 1.8 μM ), and an isomer of perillic acid. Both isomers of perillic acid, and cis and trans isomers of dihydroperillic acid were in urine hydrolysates. Intratumoral levels of d-limonene and uroterpenol exceeded the corresponding plasma levels. Other metabolites were trace constituents in tissue. Conclusions: d-Limonene is well tolerated in cancer patients at doses which may have clinical activity. The favorable toxicity profile supports further clinical evaluation. Received: 25 June 1997 / Accepted: 6 November 1997     

Plasma and CSF pharmacokinetics of ganciclovir in nonhuman primates

Purpose: The antiviral nucleoside analogue ganciclovir is a potent inhibitor of replication in herpes viruses and is effective against cytomegalovirus infections in immunocompromised patients. Ganciclovir is also used in cancer gene therapy studies that utilize the herpes simplex virus thymidine kinase gene (HSV-TK). The pharmacokinetics of ganciclovir in adults and children have been described previously but there are no detailed studies of the CNS pharmacology of ganciclovir. We studied the pharmacokinetics of ganciclovir in plasma and CSF in a nonhuman primate model that is highly predictive of the CSF penetration of drugs in humans. Methods: Ganciclovir, 10 mg/kg i.v., was administered over 30 min to three animals. Ganciclovir concentrations in plasma and CSF were measured using reverse-phase HPLC. Results: Peak plasma ganciclovir concentrations ranged from 18.3 to 20.0 μg/ml and the mean plasma AUC was 1075 ± 202 μg/ml · min. Disappearance of ganciclovir from the plasma was biexponential with a distribution half-life (t_1/2α) of 18 ± 7 min and an elimination half-life (t_1/2β) of 109 ± 7 min. Total body clearance (Cl_TB) was 9.4 ± 1.6 ml/min/kg. The mean CSF ganciclovir AUC was 168 ± 83 μg/ml · min and the mean peak CSF concentration was 0.7 ± 0.3 μg/ml. The ratio of the AUCs in CSF and plasma was 15.5 ± 7.1%. Conclusions: Ganciclovir penetrates into the CSF following i.v. administration. This finding will be useful in the design of gene therapy trials involving the HSV-TK gene followed by treatment with ganciclovir in CNS or leptomeningeal tumors. Received: 8 May 1998 / Accepted: 25 September 1998     

Four-hourly scheduling of temozolomide improves tumour growth delay but not therapeutic index in A375M melanoma xenografts

Purpose: To establish whether temozolomide is more effective against A375M human melanoma xenografts if given every 4 h rather than every 24 h, in order to exploit depletion of the DNA repair protein O ⁶-alkylguanine-DNA alkyltransferase (ATase) by prior doses of the drug. Methods: ATase depletion in A375M human melanoma xenografts was determined over 24 h after a single dose of temozolomide. The effect of different drug schedules (all of total dose 500 mg/kg) in delaying the growth of the xenografts was tested, and ATase depletion and DNA methylation damage assessed in tumour and normal tissue. Results: Maximal depletion of ATase in tumour, to 2.52 ± 0.23% of pretreatment levels, occurred 4–8 h after a single 100 mg/kg i.p. dose of temozolomide, with 23.0% recovery of protein levels at 24 h. Scheduling of temozolomide every 4 h increased tumour growth delay (33.6 ± 1.39 days with temozolomide 100 mg/kg 4-hourly ×5 versus 23.2 ± 1.43 days with temozolomide 100 mg/kg once daily ×5; P < 0.0001) at the expense of increased toxicity (17.4 ± 1.55% animal weight loss versus 10.6 ± 1.27%, respectively). Temozolomide every 4 h did not increase ATase depletion compared with the 5-day schedule, but resulted in greater DNA O ⁶-guanine methylation (29.0% more in tumour, 20.8% in liver and 56.0% in brain, comparing areas under the methylation-time curve). Conclusions: The 4-hourly schedule of temozolomide delayed tumour growth significantly more than the once-daily and 12-hourly schedules, probably as a result of greater DNA damage inflicted, but also increased toxicity. It remains to be seen if this regimen confers a net benefit over the standard schedule. Received: 12 March 1999 / Accepted: 9 August 1999     

Distribution of camptothecin after delivery as a liposome aerosol or following intramuscular injection in mice

Purpose: The plant alkaloid camptothecin (CPT) has shown significant antitumor activity against a wide variety of human tumors xenografted in nude mice. In previous studies we have found that administration of dilauroylphosphatidylcholine (DLPC) liposome aerosols containing 9-nitrocamptothecin (9-NC) inhibits the growth of human breast, colon and lung cancer xenografts. The purpose of this study was to analyze the pharmacokinetics and tissue distribution of inhaled CPT formulated in DLPC liposomes. Methods: C57BL/6 mice with subcutaneous Lewis lung carcinoma, Swiss nu/nu mice with human lung carcinoma xenografts and BALB/c mice without tumors were used for pharmacokinetic studies of CPT administered as a liposome aerosol and BALB/c mice were given CPT intramuscularly. Results: After 30 min inhalation of CPT liposome aerosol, drug was deposited in the lungs (310 ng/g) and was followed promptly by the appearance of high concentrations in the liver (192 ng/g) and with lesser amounts appearing in other organs. Drug concentration in the brain was 61 ng/g. After intramuscular injection of CPT dissolved in DMSO, drug was released from the site of injection very slowly and accumulated mainly in the liver (136 ng/g). Only trace amounts appeared in the lungs (2–4 ng/g). These results demonstrate a prompt pulmonary and later systemic distribution of CPT following liposome aerosol administration. Conclusions: The substantial concentrations of CPT in lungs and other organs following inhalation of liposome aerosol suggest the possible benefit of it and of its more active derivative, 9-NC, in the treatment of lung, liver, kidney and brain cancer in humans. Received: 23 September 1998 / Accepted: 11 January 1999     

Reduced Mobilisation of Hematopoietic Stem Cells After Hepatic Resection for Malignant Liver Disease

Recent studies have demonstrated that hematopoietic stem cells (HSCs) can mobilize following liver resection, thus contributing to the repair of hepatic damage. Aim of this study has been to determine whether the nature of the hepatic lesion (benign vs. malignant disease) can give rise to a different degree of mobilisation of HSCs. Two groups of patients were selected: the first included seven patients undergoing hepatic resection (five major and two minor) for a benign liver disease (focal nodular hyperplasia, hemangioma cavernosa, angioma, biliary adenofibroma) and the second included seven patients undergoing hepatic resection (five major and two minor) for a malignant (either primary or secondary) liver disease. White blood cell count and CD34+ (percentage and total number) at time T₀ (basal value before surgery) and at time T₁ (value on the sixth–eighth day after surgery) have been evaluated by standard methods. In the group undergoing hepatic resection for a benign liver disease, a significant increase of CD34+ cells, both in percentage (0.082 ± 0.043 vs. 0.048 ± 0,026, p = 0.041) and in absolute number (8.14 ± 5.95 vs. 3.26 ± 2.63, p = 0.018) have been documented, as opposed to the group of patients affected with a malignant liver disease, where no significant variation has been observed (CD34+ %: 0.044 ± 0.033 vs. 0.041 ± 0.031, p: n.s.; CD34+ total number: 3.52 ± 2.56 vs. 2.27 ± 2.01, p = n.s.) These results show a different bone marrow response to the surgical liver resection depending on the nature of the lesion, thus emphasizing a reduced mobilisation of HSCs in the malignant diseases. Since it has been documented that the type of the hepatic lesion can induce a different regenerative response, it has to be explained how the neoplastic lesions can negatively influence the mobilization of HSCs. It can be hypothesized that a variety of humoral factors, including stromal cell-derived factor, matrix metalloproteinases, hepatocyte growth factor and interleukin-8 can influence the process of mobilization of HSCs after liver resection surgery. These substances are also involved in the mechanisms of development and metastasising of many tumours. It is probably in this context that a reason may be found for the different mobilisation of hematopoietic stem cells, depending on the nature of the hepatic lesion treated, that was encountered in this study.     

Metabolism and biliary excretion of trimetrexate by the isolated perfused rat liver

The metabolism and biliary excretion of trimetrexate (TMTX), a lipid soluble antifolate, were examined using a recirculating isolated perfused rat liver system. Elimination of TMTX into perfusate was biphasic and dose-independent, with distribution and elimination half-lives of 2 and 13 min. Two metabolites, M1 and M2, both known to inhibit dihydrofolate reductase activity, were present in perfusate only in small concentrations. However, of the total TMTX dose, approximately 50% was excreted in bile as M1, and 20% as M2. Up to 75% of the total dose was accounted for as TMTX, M1, or M2 in perfusate and bile.