Pharmacologic effects of paclitaxel in human bladder tumors

Purpose: The goal of this study was to determine whether paclitaxel, when given by a 2-h treatment, produces significant cytotoxic effects in human bladder transitional cell carcinoma and hence qualifies as a candidate drug for intravesical treatment. Methods: Histocultures of surgical specimens from patients (n = 16) were used. Results: Paclitaxel produced partial inhibition of DNA precursor incorporation in about 70% of tumors and induced apoptosis in about 90% of tumors, while these effects were minimal or not detectable in the remaining tumors. In the responsive tumors, the average maximal inhibition of DNA synthesis was 60% and the average maximal apoptotic index was 15%. Resistance to antiproliferative and apoptotic effects was not always found in the same individual tumors, and no relationship was found between the magnitude of antiproliferative and apoptotic effects in individual tumors. The maximal apoptotic index correlated with the LI for the untreated control (r ² = 0.42, P < 0.01). More than 95% of apoptotic cells were labeled by DNA precursor, whereas not all labeled cells were apoptotic. The pharmacologic effects of paclitaxel in bladder tumors were qualitatively equivalent to those previously found in human head and neck tumors and in human prostate tumors after treatment for longer periods of 24 to 96 h. Conclusions: These results indicate that a 2-h paclitaxel treatment was sufficient to produce antiproliferation and apoptosis in 70–90% of human bladder tumors, and the apoptotic effect appeared to be linked to proliferation and occurred after DNA synthesis. Received: 19 December 1996 / Accepted: 20 March 1997     

A phase I evaluation of multitargeted antifolate (MTA, LY231514), administered every 21 days, utilizing the modified continual reassessment method for dose escalation

Purpose: To determine toxicities, maximally tolerated dose (MTD), pharmacokinetic profile, and potential antitumor activity of MTA, a novel antifolate compound which inhibits the enzymes thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFT), and dihydrofolate reductase (DHFR). Methods: Patients with advanced solid tumors were given MTA intravenously over 10 min every 21 days. Dose escalation was based on the modified continual reassessment method (MCRM), with one patient treated at each minimally toxic dose level. Pharmacokinetic studies were performed in all patients. Results: A total of 37 patients (27 males, 10 females, median age 59 years, median performance status 90%) were treated with 132 courses at nine dose levels, ranging from 50 to 700 mg/m². The MTD of MTA was 600 mg/m², with neutropenia and thrombocytopenia, and cumulative fatigue as the dose-limiting toxicities. Hematologic toxicity correlated with renal function and mild reversible renal dysfunction was observed in multiple patients. Other nonhematologic toxicities observed included mild to moderate fatigue, anorexia, nausea, diarrhea, mucositis, rash, and reversible hepatic transaminase elevations. Three patients expired due to drug-related complications. Pharmacokinetic analysis during the first course of treatment at the 600 mg/m² dose level demonstrated a mean harmonic half-life, maximum plasma concentration (Cpmax), clearance (CL), area under the curve (AUC), and apparent volume of distribution at steady state (Vdss) of 3.08 h, 137 μg/ml, 40.0 ml/min per m², 266 μg · h/ml, and 7.0 l/m², respectively. An average of 78% of the compound was excreted unchanged in the urine. Partial responses were achieved in two patients with advanced pancreatic cancer and in two patients with advanced colorectal cancer. Minor responses were obtained in six patients with advanced colorectal cancer. Conclusions: The MTD and dose for phase II clinical trials of MTA when administered intravenously over 10 min every 21 days was 600 mg/m². MTA is a promising new anticancer agent. Received: 20 October 1998 / Accepted: 30 March 1999     

Pharmacokinetics of paclitaxel administered in combination with cisplatin, etoposide and bleomycin in patients with advanced solid tumours

 Purpose: To evaluate the pharmacokinetics of paclitaxel and cisplatin administered in combination with bleomycin and etoposide and Granulocyte Colony-Stimulating Factor (G-CSF) in patients with advanced solid tumours. Methods: Patients were recruited to a phase I trial where escalating doses of paclitaxel (125 to 200 mg/m²) were administered in combination with etoposide 100 or 120 mg/m², and fixed dose of cisplatin 20 mg/m² and bleomycin 30 mg, with the concomitant use of G-CSF. Paclitaxel (3-h infusion) was followed by 1-h etoposide, 4-h cisplatin and 30-min bleomycin infusions, respectively. Pharmacokinetics sampling for paclitaxel analysis was performed in ten patients from dose levels II–V. Results: The mean paclitaxel area under the plasma concentration-versus-time curves (AUC) for the 125-mg/m² dose level (II) was 7.0 ± 3.6 h μmol⁻¹ l⁻¹, for the 175-mg/m² dose level (III) 10.6 ± 2.8 h μmol⁻¹ l⁻¹, for the 200-mg/m² dose level (IV) it was 16.0 ± 5.0 h μmol⁻¹ l⁻¹, and for the 175-mg/m² dose level (V) it was 12.5 ± 6.1 h μmol⁻¹ l⁻¹. The mean peak plasma concentration (C_max) values for dose levels II–V were 1.9 ± 1.1 μmol/l, 3.4 ± 1.2 μmol/l, 4.3 ± 1.0 μmol/l and 3.8 ± 1.2 h μmol/l, respectively. Conclusion: In this study, relevant pharmacokinetic parameters of paclitaxel like AUC, C_max and the paclitaxel plasma concentration above the pharmacologically relevant 0.1-μmol/l threshold concentration (t > 0.1 μM) when administered in combination with cisplatin, etoposide and bleomycin (PEB) were not statistically different from paclitaxel data of historical controls. However, given the trial design, pharmacokinetic interactions between the agents cannot be excluded. Received: 29 June 1998 / Accepted: 29 January 1999     

Investigation of bioavailability and pharmacokinetics of treosulfan capsules in patients with relapsed ovarian cancer

Purpose: Treosulfan (l-threitol-1,4-bis-methanesulfonate, Ovastat) is a prodrug of a bifunctional alkylating agent with activity in ovarian carcinoma and other solid tumors. In a pharmacologic study of the bioavailability of treosulfan in a capsule formulation, patients with relapsed ovarian carcinoma were treated with alternating doses of oral and intravenous (i.v.) treosulfan of 1.5 or 2.0 g daily for 5 to 8 days. Methods: A sensitive method for the determination of treosulfan in plasma and urine by reversed-phase high-performance liquid chromatography had previously been developed. Pharmacokinetic analyses of treosulfan were carried on plasma and urine samples from 20 i.v. courses and 20 courses of oral administration. Results: The bioavailability ratio (f) of oral to i.v. administration was calculated as 0.97 ± 0.18 (mean ± SD) using the values AUC_oral=82.1 ± 39.4 μg/ml h and AUC_i.v.=85.4 ± 30.3 μg/ml h. The peak plasma concentration c_max (29 ± 14 μg/ml vs 65 ± 23 μg/ml) was significantly (P < 0.01) higher after i.v. administration and the t_max after oral administration was 1.5 ± 0.34 h. The terminal half-life of treosulfan was about 1.8 h. The mean urinary excretion of the parent compound was about 15% of the administered total dose over 24 h (range 6–26%). Conclusions: The high and relatively constant bioavailability of treosulfan indicates that capsules provide a satisfactory noninvasive treatment alternative. A feasible and reliable oral treosulfan formulation could provide the basis for the development of long-term low-dose outpatient treatment of patients with malignant diseases. Received: 28 July 1999 / Accepted: 16 December 1999     

Cremophor reduces paclitaxel penetration into bladder wall during intravesical treatment

Purpose: We have previously shown that paclitaxel, when dissolved in water and instilled into the bladder, readily penetrates the urothelium. The FDA-approved formulation uses Cremophor and ethanol to dissolve paclitaxel. In the present study, the effects of this solvent system on the urine, bladder tissue, and plasma pharmacokinetics of intravesical paclitaxel were evaluated. Methods: Plasma, urine, and tissue pharmacokinetics were determined in five dogs treated for 120 min with paclitaxel (500 μg per 20 ml of 0.22% w/v Cremophor and 0.21% v/v ethanol) by intravesical instillation. Equilibrium dialysis was used to determine the free fraction of paclitaxel and the presence of Cremophor micelles was verified using a fluorescent probe method. Results: The average bladder tissue concentration was >1600-fold higher than the plasma concentration. Comparison of the results for paclitaxel dissolved in Cremophor/ethanol with our previous results of paclitaxel dissolved in water (500 μg per 20 ml) indicates that Cremophor/ethanol decreased the paclitaxel partition across the urothelium and reduced the average bladder tissue concentration by 75%, but did not alter the rate of paclitaxel penetration across the bladder wall, the urine pharmacokinetics or the plasma pharmacokinetics of paclitaxel. For Cremophor, the urine concentrations during the 120-min treatment ranged from 0.12% to 0.22%, and the concentration in bladder tissue from 0.00004% to 0.0009%. The threshold Cremophor concentration for micelle formation was 0.008%. We found that ethanol at concentrations up to 1% and Cremophor at concentrations below 0.01% did not alter the free fraction of paclitaxel, whereas Cremophor at higher concentrations, i.e. 0.065% and 0.25%, significantly reduced the free fraction by two- to six-fold, respectively. These results indicate that during intravesical instillation of the FDA-approved paclitaxel formulation, the concentration of Cremophor in urine was sufficient to form micelles, resulting in sequestration of paclitaxel into micelles, reduction in the free fraction of paclitaxel and consequently a reduction in paclitaxel penetration across the urothelium. In contrast, the Cremophor concentrations in bladder tissue were inadequate to form micelles and thus did not alter the drug penetration through the bladder tissue. Conclusions: We conclude that intravesical paclitaxel treatment using the FDA-approved formulation provides a significant bladder tissue targeting advantage, although the advantage is lower than when paclitaxel is dissolved in water. Received: 1 October 1998 / Accepted: 12 January 1999     

Clinical pharmacokinetics of intravenous treosulfan in patients with advanced solid tumors

Treosulfan (l-threitol-1,4-bis-methanesulfonate, Ovastat) is a prodrug of a bifunctional alkylating agent with activity in ovarian carcinoma and other solid tumors. For a clinical and pharmacology study, patients with advanced, refractory, or resistant solid tumors were treated with a single-dose intravenous 30-min infusion of 8 or 10 g/m² treosulfan. A sensitive method for the determination of treosulfan in plasma and urine by reverse-phase high-performance liquid chromatography was developed. A total of 14 plasma and urine treosulfan pharmacokinetics determinations were analyzed in the 8-g/m² group and 7 were analyzed in the 10-g/m² group, the maximum tolerated dose for this group of pretreated patients. The terminal half-life of treosulfan was in the range of 1.8 h. AUC and C_max values were significantly (P < 0.01) higher in the 10-g/m² group (AUC 708 ± 168 versus 977 ± 182 μg ml⁻¹ h, C_max 465 ± 98 versus 597 ± 94 μg/ml). The mean urinary excretion of the parent compound was about 25% of the total dose delivered over 48 h (range 5–49%), and about 20% was excreted during the first 6 h after administration. Currently, a clinical phase I pharmacokinetics and dose-escalation trial with autologous blood stem-cell support has been started at 20 g/m² treosulfan using a 2-h infusion protocol. Received: 25 August 1997 / Accepted: 15 December 1997     

The significance of the sequence of administration of topotecan and etoposide

 Purpose: Often the best method of integrating chemotherapeutic agents is unknown. Recently there has been interest in the use of combinations of the topoisomerase II inhibitors and the topoisomerase I inhibitors as these agents have shown individual activity in malignancies such as non-small-cell lung cancer. This study examined the interaction of the topoisomerase II inhibitor etoposide with the topoisomerase I inhibitor topotecan (Tpt) in V79 cells (hamster lung fibroblast cells) to determine the optimal method of delivering these agents. Methods and results: Cell survival was assessed by colony formation. Synergistic interactions were assessed by the median effect principle in which a combination index (CI) of less than one suggests a synergistic interaction. The V79 cells were exposed to sequential 24-h incubations with the two chemotherapeutic agents. Initially, equitoxic doses of the two agents were delivered (i.e. 0.0275 μg/ml of topotecan alone or 0.089 μg/ml of etoposide alone resulting in a surviving fraction of 70%; Tpt : etoposide ratio 1 : 3.2). It was determined that a sequence-dependent synergistic interaction (CI<1) resulted at a lower level of cytotoxicity if the etoposide exposure followed the Tpt exposure compared to the opposite sequence. This same effect was seen after treatment of cells with various concentration (μg/ml) ratios of Tpt : etoposide (1 : 4.0, 1 : 1, 2.5 : 1). Conclusions: These results suggest that maximum synergy occurs for the delivery of etoposide following Tpt exposure (compared to the opposite sequence) and these findings may have important clinical implications. Received: 29 September 1995/Accepted: 25 March 1996     

Multiple factors other than p53 influence colon cancer sensitivity to paclitaxel

Purpose: To determine factors which influence the sensitivity of human colorectal carcinoma cell lines to paclitaxel. Methods: The paclitaxel sensitivity of ten human colorectal carcinoma cell lines, and a panel of RKO colon carcinoma cell lines, isogenic except for p53 status, were studied. The inhibitory concentrations causing a 50% decrease in growth (IC₅₀) were assayed after 3, 24, and 96 h after paclitaxel exposure. The doubling time (DT) and cell cycle parameters of cells were also measured. The expression of the multidrug resistance glycoprotein-1 (MDR-1), bcl-2 and bax was quantitatively assessed by immunoblotting. Results: Mean IC₅₀ values at 24 and 96 h drug exposure were about 1.5 logs lower than the IC₅₀ values at 3 h, regardless of the p53 status. No difference was found between the IC₅₀ values of wild-type and mutant p53 cells, or among the RKO panel of cells. Correlation analysis showed that: (1) resistance was associated with longer DTs, but this was generally abated by a 96-h exposure; (2) with a 3-h exposure, the combination of MDR, bcl-2 and bax parameters with DT (DT + MDR + bcl-2–bax) best correlated with IC₅₀ values (r=0.77); (3) with a 96-h exposure, in spite of the generally decreased IC₅₀ values, a combination of MDR-1, bcl-2 and bax parameters (MDR + bcl-2–bax) best correlated with the IC₅₀ values (r=0.71). Conclusions: These results suggest that the exposure duration, DT, and expression of MDR-1, bcl-2 and bax each contribute to paclitaxel sensitivity of human colorectal carcinoma cells. In assessing paclitaxel drug resistance, multiple factors should always be considered. There may be a therapeutic window for taxanes in colon cancer by optimizing pharmacokinetics and modulating MDR-1 and bcl-2 resistance factors. Received: 13 September 1999 / Accepted: 26 April 2000     

Primary treatment of low-grade non-Hodgkin's lymphoma using an all oral anthracycline-containing regimen, chlorambucil, idarubicin, dexamethasone (CID) – a phase II study

Purpose: The alkylating cytostatic prodrug cyclophosphamide is bioactivated by the human cytochrome P450 enzyme system. Since these enzymes are not only expressed in human liver, but also in extrahepatic tissue, local bioactivation of this drug may play an important role in its antineoplastic effects, e.g., chemotherapy of lung tumors. This would require uptake of significant amounts of cyclophosphamide into tumor tissue, which has not yet been demonstrated. Methods: We used a recently developed, ex vivo isolated, ventilated and perfused human lung model to study cyclophosphamide uptake into bronchial carcinoma and healthy lung tissue. Following a standard lobectomy, lung samples containing the tumor were perfused with buffer containing 2 mM cyclophosphamide for 2 h. Cyclophosphamide concentrations in perfusate and healthy peripheral tissue were measured during the perfusion and in tumors at the end of perfusion. Results: In all tissue samples, cyclophosphamide uptake was relatively poor, indicated by a tissue to perfusate ratio of 0.021. Moreover, in tumor samples, cyclophosphamide concentrations were significantly lower (P < 0.05) than in healthy lung tissue and showed pronounced interindividual variability. Median concentrations were 36.8 μg/g (26.9–44.2 μg/g) in healthy tissue and 5.1 μg/g (0.0–26.8 μg/g) in tumor samples. Tumor cyclophosphamide concentrations varied between 0 and 75% of those reached in healthy tissue. Conclusions: Our results indicate that CP tumor concentrations are modulated by factors different from dose and that expression of bioactivating enzymes in human lung or transfection of genes encoding these enzymes into tumor cells does not necessarily lead to local bioactivation of cyclophosphamide. Received: 4 March 1999 / Accepted: 8 June 1999     

Increased 9-aminocamptothecin dose requirements in patients on anticonvulsants

Background: High grade astrocytomas remain uniformly fatal despite aggressive surgery and radiotherapy. As existing chemotherapeutic agents are of limited benefit, clinical trials are underway to screen new drugs, such as 9-aminocamptothecin (9-AC), for activity in high grade astrocytomas. Purpose: This study was designed to estimate the efficacy of 9-AC in patients with newly diagnosed glioblastoma multiforme and recurrent high grade astrocytomas. The planned dose of 9-AC for this trial was 850 μg/m² per 24 h as a 72-h continuous intravenous infusion every 2 weeks. This was the maximum tolerated dose (MTD) on this schedule in multiple phase I studies in patients with systemic malignancies. However, we found this dose subtherapeutic in our patient population. As a result, the purpose of the study was altered to determine the MTD. Methods: A group of 32 patients were studied using 850 μg/m² per 24 h with a provision to escalate to 1000 μg/m² per 24 h if the first three cycles of 9-AC were without significant hematologic toxicity. Once it was determined that myelosuppression did not occur in patients on anticonvulsants, dose escalations were initiated using the continual reassessment method. Dose escalations were conducted independently in newly diagnosed and recurrent patients and in those taking and not taking hepatic enzyme-inducing anticonvulsants. Pharmacologic studies were conducted during the first cycle of 9-AC. Toxicity was determined using the NCI common toxicity criteria and efficacy was assessed using serial volumetric brain scans. Results: 9-AC was administered to 59 patients, 31 with newly diagnosed glioblastoma multiforme and 28 with recurrent high grade astrocytomas. No grade III–IV myelosuppression was noted in the 29 patients (128 cycles) on phenytoin, carbamazepine, phenobarbital, and/or valproic acid who received 850 μg/m² per 24 h. In contrast, two of three patients (five cycles) who were not taking anticonvulsants developed grade IV myelosuppression. Steady-state total 9-AC plasma levels were lower in patients on anticonvulsants (median 25.3 nM ) than in patients who were not taking anticonvulsants (median 76.5 nM ). Dose escalations performed in 27 additional patients determined the MTD in patients taking anticonvulsants to be 1776 μg/m² per 24 h for patients with newly diagnosed tumors and 1611 μg/m² per 24 h for patients with recurrent disease. Conclusions: We describe a new and unexpected drug interaction between 9-AC and anticonvulsants. This is similar to recent findings with paclitaxel, and suggests that higher than “usual” doses of some chemotherapeutic agents are required in patients on anticonvulsants. Prospectively defined dose escalations and pharmacologic studies are essential for the careful evaluation of new chemotherapeutic agents in patients with brain tumors. Received: 21 April 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     

Dose escalation and pharmacokinetic study of irinotecan in combination with paclitaxel in patients with advanced cancer

Purpose: Based on preclinical data demonstrating synergy between camptothecin analogues and taxanes, we determined the maximum tolerated dose (MTD) of irinotecan that could be given in combination with a fixed dose of paclitaxel of 75 mg/m², when both drugs were delivered on a weekly schedule. The pharmacokinetics of this combination were explored to determine whether the sequence of administration affected the elimination of irinotecan. Methods: For the first cycle patients with advanced cancer were treated with irinotecan given as a 90-min infusion followed immediately by paclitaxel given at a dose of 75 mg/m² over 1 h. The sequence of drug administration was reversed in subsequent cycles for most patients. Chemotherapy was given weekly for 4 weeks, followed by a 2-week rest. In selected patients, plasma concentrations of irinotecan were determined by high-performance liquid chromatography during the first 24 h of cycle 1 and after the first dose of cycle 2 to determine whether the order of drug administration affected the elimination of irinotecan, or the toxicologic effects of the chemotherapy. Results: A total of 53 cycles were delivered to 21 patients. Reversible neutropenia was dose-limiting. Suppression of the other blood cell elements was modest. There was one partial response in a man with a previously treated cholangiocarcinoma that lasted 26 weeks. Prolonged stabilization of disease (6 months or more) was observed in five of the patients (24%). At the recommended dose of irinotecan (50 mg/m²), transfusions of red cells and platelets were not required. The sequence of drug administration produced no significant differences in the pharmacokinetic parameters of irinotecan or SN-38, which were similar to the values reported when irinotecan is administered alone. The most prominent nonhematologic toxicities were mild diarrhea and fatigue. Conclusions: The recommended dose of irinotecan on this schedule is 50 mg/m². The sequence of drug administration affects neither the elimination of irinotecan nor the chemotherapy-related toxicity. This combination is well tolerated and causes minimal clinical side effects. Received: 5 July 1999 / Accepted: 3 February 2000     

Pharmacokinetics of oxaliplatin in patients with normal versus impaired renal function

Purpose: The pharmacokinetics (PK) of platinum was investigated and compared in patients with normal (NRF) and impaired renal function (IRF), after they had received oxaliplatin at the recommended dose and delivery modality. Methods: Oxaliplatin was administered at 130 mg/m² as a 2-h infusion without hydration. Patients were recruited and classified according to their creatinine clearance (CrCl > or < 60 ml/min), calculated using the Cockcroft and Gault formula. Blood was taken for PK analysis during and after the infusion. Twenty-three patients were included in the PK analysis (13 NRF and 10 IRF). At inclusion, the median CrCls were 70.5 ml/min (range 63–136) for the NRF group and 42 ml/min (range 27–57) for the IRF group. Three patients underwent a second course of treatment and additional blood sampling for analysis. Platinum levels in the plasma, ultrafiltrate and red blood cells (RBCs) were measured using flameless atomic absorption spectrophotometry (FAAS). Results: Following the administration of oxaliplatin, platinum binding to plasma proteins and RBCs was rapid and extensive; at the end of the 2-h infusion, 27% of the platinum in the plasma remained free (40% bound to RBCs, 33% bound to plasma proteins). Neither the mean maximal concentration (C_max) of total platinum in the plasma, the mean C_max of ultrafilterable platinum in the plasma, nor the maximal platinum content in the RBCs differed significantly between the two groups (2.59 vs 2.58 μg/ml, 1.09 vs 1.28 μg/ml and 2.06 vs 2.17 μg/ml, respectively, for patients with NRF vs IRF). After the end of the infusion, levels of total and free (ultrafilterable) platinum in the plasma declined biexponentially. The plasma clearance of both total and free platinum as well as the area under the curve (AUC) of the free platinum fraction correlate with the calculated CrCl (P=9 × 10⁻³, P=3.1 × 10⁻⁵ and P=9 × 10⁻⁶, respectively). After a single course of oxaliplatin, toxicities reported in the two groups of patients were similar. Conclusions: Our results are in agreement with the in vitro data concerning the extensive binding of oxaliplatin to plasma proteins and RBCs. They also reveal a strong negative correlation between free drug plasma availability and renal function, with a corresponding positive correlation between clearance of the plasmatic platinum and renal function. Thus, renal impairment entails a greater overall exposure to platinum in the plasma. However, this study failed to elicit any relationship between moderate renal impairment and the acute toxicity associated with oxaliplatin. Received: 8 April 1999 / Accepted: 30 July 1999     

Phase I/II study of cisplatin, ifosfamide and irinotecan with rhG-CSF support in patients with stage IIIB and IV non-small-cell lung cancer

Purpose: We conducted a phase I/II study in previously untreated patients with stage IIIB or IV non-small-cell lung cancer (NSCLC) to: (1) determine the maximum tolerated dose (MTD) of cisplatin combined with a fixed schedule of ifosfamide and irinotecan with rhG-CSF support; and (2) to determine the overall response rate and median survival of patients entered on this study. Methods: Ifosfamide (1.5 g/m²) and irinotecan (60 mg/m²) were administered at fixed doses on days 1–4 and on days 1, 8 and 15, respectively. Cisplatin was given on day 1 at 60 mg/m² and was increased in 10-mg/m² increments. This regimen was repeated every 4 weeks. rhG-CSF (nartograstim) was administered subcutaneously at a dose of 1 μg/kg on days 5–18 except on the day of irinotecan treatment. Results: Between June 1995 and April 1998, 46 patients were registered onto this phase I/II study. The MTD of cisplatin was defined according to toxicity and the dose during three courses was increased. Since at the 80 mg/m² dose level more than one-third of the patients were treated with dose modification, the dose of 70 mg/m² was recommended for phase II study. The dose-limiting toxicity was leukopenia. The overall response rate was 62.2% (95% CI 48.0–76.4%), the median response duration was 144 days, and the median survival time was 393 days. Conclusion: For phase II study, we recommend doses of cisplatin 70 mg/m² on day 1 combined with ifosfamide and irinotecan with rhG-CSF support. Both the response rate and preliminary survival data in this study suggest a high degree of activity of this combination in previously untreated NSCLC. Received: 29 April 1999 / Accepted: 15 September 1999     

Cytostatic and apoptotic effects of paclitaxel in human breast tumors

Purpose: We have previously reported incomplete cytotoxic responses of other human solid tumors (bladder, head and neck, ovarian and prostate) to paclitaxel. This finding is qualitatively different from the nearly complete response observed in monolayer cultures of human cancer cell lines. The present study examined the pharmacodynamics of paclitaxel in human breast tumors. Methods: Three-dimensional histocultures of patient tumors were used. The cytostatic effect was evaluated by measurement of the inhibition of 48-h cumulative bromodeoxyuridine (BrdUrd) incorporation. The apoptotic effect was evaluated in terms of morphological changes and by in situ DNA end labeling. Results: Paclitaxel produced partial cytostasis (∼30% maximum) and induced apoptosis (maximum apoptotic index of 3.3% to 29%) in all 15 tumors. More than 95% of apoptotic cells were BrdUrd labeled, but not all BrdUrd-labeled cells were apoptotic. The maximal apoptotic indices in the tumors were significantly correlated with the BrdUrd labeling index of untreated controls (r ² = 0.63, P < 0.01). The maximum apoptotic effect was observed at a tenfold lower drug concentration (0.1 μM ) compared to the maximum cytostatic effect (1 μM ). Neither of these effects was enhanced by increasing the drug concentration to 10 μM. Conclusions: The pharmacodynamics of paclitaxel in human breast tumors are comparable to those found in other human solid tumors. The labeling of apoptotic cells by BrdUrd and the correlation between the proliferation index and apoptosis suggest that drug-induced apoptosis is linked to cell proliferation and is completed after DNA synthesis. The finding that maximal cytostatic and apoptotic effects of paclitaxel were achieved at or below the clinically achievable concentration of 1 μM suggests further increasing the dose to elevate plasma concentration beyond 1 μM may not improve treatment outcome. Received: 3 July 1997 / Accepted: 6 November 1997     

Pharmacokinetic analysis of high-dose toremifene in combination with doxorubicin

Purpose: Toremifene (Fareston) is an orally administered triphenylethylene derivative with chemosensitizing activity in vitro in estrogen receptor-negative multidrug-resistant human breast cancer cells. The purpose of this study was to evaluate the effects of high-dose toremifene (600 mg/day for 5 days) on the plasma pharmacokinetics of doxorubicin in humans. The 600-mg dose had been previously established as the maximum tolerated dose in a phase I study of 35 patients. Methods: Doxorubicin was administered as an intravenous (i.v.) bolus over 15 min at a dose of 60 mg/m² to 11 patients in the absence of toremifene pretreatment to establish baseline doxorubicin pharmacokinetics. Six of these patients received 600 mg/day toremifene for 5 days 4 weeks later, followed by an i.v. bolus dose of doxorubicin (60 mg/m²) on day 5. During toremifene pretreatment, blood specimens (5 ml) were drawn at 0, 2, 4, and 24 h after dosing to assess peak levels. Following doxorubicin administration in each cycle, blood specimens were collected over a 72-h period for determination of the terminal half-life of elimination. Plasma concentrations of doxorubicin and toremifene were assessed by high-performance liquid chromatography (HPLC). Cumulative linear areas under the time-concentration curve (AUC) for doxorubicin were calculated using a noncompartmental model. Results: Prior to toremifene dosing, baseline doxorubicin pharmacokinetic studies showed an average terminal half-life of elimination of 40.04 ± 7.86 h in 4 patients, and an average AUC of 135 600 ± 67 600 μg/ml · h in 11 patients. In 4 of the patients receiving 600 mg/day toremifene for 5 days, the average terminal half-life of elimination was 38.12 ± 7.81 h, and the average AUC was 141 900 ± 62 900 μg/ml · h in 6 patients, i.e. a slight increase of 4.6%. No statistically significant change in the doxorubicin elimination kinetics with or without toremifene therapy was observed. Conclusions: Toremifene does not appear to interfere with the elimination kinetics of doxorubicin. Received: 1 July 1997 / Accepted: 16 December 1997     

Clinical significance of <Emphasis Type="Italic">Maspin</Emphasis> promoter methylation and loss of its protein expression in invasive ductal breast carcinoma: correlation with VEGF-A and MTA1 expression

Maspin is a serine protease inhibitor with tumor-suppressor activity. Maspin can suppress tumor growth and metastasis in vivo and tumor cell motility and invasion in vitro. Previous studies indicate that the loss of Maspin expression is closely linked to aberrant methylation of the Maspin promoter. We examined the promoter methylation status of Maspin in tumor and corresponding serum of breast cancer patients. In addition, protein expression of this gene was also assessed to determine possible correlation between promoter hypermethylation and gene silencing. Further, we investigated the correlation of Maspin expression with vascular endothelial growth factor (VEGF-A) and MTA1 expression. Maspin methylation was analyzed by methylation-specific PCR in 100 invasive ductal breast carcinoma patients’ tumors and circulating DNA in a prospective study. Promoter hypermethylation was correlated with expression of the encoded protein in tumors by immunohistochemistry. Significant correlation was observed between promoter hypermethylation of Maspin (r = +0.88; p ≤ 0.0001) in tumors and paired sera. Significant association was found between Maspin promoter hypermethylation and loss of its protein expression (p = 0.01, OR = 3.1, 95% CI = 1.3–7.4). The expression of VEGF-A and MTA1 was lower in tumors with high Maspin expression compared to tumors with loss of Maspin expression. Our results indicate that aberrant promoter methylation is associated with loss of Maspin immunoreactivity in breast cancer tissues. Further, loss of Maspin expression is significantly correlated with increased expression of VEGF-A and MTA1.     

Pharmacokinetics of oral O6-benzylguanine and evidence of interaction with oral ketoconazole in the rat

Purpose: O⁶-Benzylguanine (BG) is a modulator of the DNA repair protein, O⁶-alkylguanine-DNA alkyltransferase (AGT). BG is converted in mice, rats and humans to an equally active, yet longer-lived metabolite, O⁶-benzyl-8-oxoguanine (8-oxo-BG) by CYP1A2, CYP3A4 and aldehyde oxidase. Since intravenous BG is expected to enter phase I development with orally administered anticancer agents such as temozolomide, procarbazine or SarCNU, we determined the bioavailability of orally administered BG, as well as the effect of ketoconazole, a potent intestinal and hepatic CYP3A4 inhibitor, on the disposition of BG. Methods: Following intravenous or oral administration of BG in PEG-400/saline (40:60) to Sprague-Dawley rats, the pharmacokinetics of BG and 8-oxo-BG were determined. To determine the effect of CYP3A inhibition on disposition, oral BG was coadministered with ketoconazole. Results: The peak plasma concentration (C_max), time to C_max (t_max), and bioavailability (F) of oral BG were: 2.3 ± 0.9 μg/ml, 2.3 ± 0.6 h, and 65.5% respectively. The AUCs of BG and 8-oxo-BG were 13.1 ± 4.6 μg · h/ml and 1.7 ± 0.4 μg · h/ml after oral administration of BG. Coadministration with ketoconazole resulted in an increase in mean absorption time from 2.0 ± 0.3 h to 6.0 ± 0.9 h, a shift in t_max to 5 ± 3.3 h, a decrease in C_max to 0.96 ± 0.8 μg/ml, and a decrease in AUC_0-inf ratio of 8-oxo-BG:BG from about 0.12 to 0.04 (P < 0.05). The bioavailability of BG was not changed (65.5% vs 56.9%, P=0.78). Conclusions: The oral bioavailability of BG is high, warranting consideration of an oral formulation for clinical development. Coadministration of ketoconazole and BG resulted in delayed oral absorption and inhibition of conversion of BG to 8-oxo-BG in the rat model. Received: 12 July 1999 / Accepted: 11 February 2000     

A phase II/pharmacokinetic trial of high-dose progesterone in combination with paclitaxel

Purpose: The purpose of this study was to investigate the effect of high-dose progesterone, an inhibitor of P glycoprotein, on the pharmacokinetics and toxicity of paclitaxel. Patients and methods: A total of 29 patients with various tumors were treated with single-agent paclitaxel (125 mg/m² administered over 3 h once every 3 weeks) until progression of disease, at which point high-dose progesterone (3 g administered i.v. over 24 h) was added to the paclitaxel treatment program in 20 patients (13 women, 7 men). Pharmacokinetic studies of paclitaxel administered alone and with progesterone were performed in eight patients. Results: The pharmacokinetic parameters of paclitaxel were highly variable. High-dose progesterone increased the peak plasma levels (3.00 ± 0.94 vs. 4.15 ± 1.63 M; P=0.029; mean ± SD) and the area under the curve (AUC; 14.3 ± 4.75 vs. 17.3 ± 5.59 M × h; P=0.006) of paclitaxel. The absolute neutrophil and platelet nadir counts did not differ significantly between the paclitaxel and the combined treatment cycles. Three of the 20 patients documented to have progressive disease on paclitaxel alone had partial responses when high-dose progesterone was added to the paclitaxel regimen. Conclusion: Progesterone had a statistically significant impact on the pharmacokinetics of paclitaxel. The addition of high-dose progesterone to paclitaxel is feasible, but the small number of patients prevents conclusions being drawn about the clinical efficacy of combined progesterone and paclitaxel. Received: 11 September 1998 / Accepted: 15 February 1999     

In vitro schedule-dependent interaction between paclitaxel and SN-38 (the active metabolite of irinotecan) in human carcinoma cell lines

Paclitaxel and irinotecan are important new anticancer agents. The combination of these two agents has been considered for use against a variety of advanced solid tumors. Since the schedule-dependent effects of this combination may be crucial to its use, we studied the interaction of paclitaxel and SN-38 (the active metabolite of irinotecan) in various schedules in four human cancer cell lines in culture. Cell growth inhibition after 5 days was determined using an MTT assay. The effects of drug combinations at the IC₈₀ level were analyzed by the isobologram method. Simultaneous exposure to paclitaxel and SN-38 for 24 h produced antagonistic (subadditive and protective) effects in the human lung cancer cell line A549, the breast cancer cell line MCF7, and the colon cancer cell line WiDr, and produced additive effects in the ovarian cancer cell line PA1. Sequential exposure to paclitaxel for 24 h followed by SN-38 for 24 h, and the reverse sequence, produced additive effects in all four cell lines. These findings suggest that sequential administration, not simultaneous administration, may be the appropriate schedule for the therapeutic combination of paclitaxel and irinotecan. Continued preclinical and clinical studies should provide further insights and assist in determining the optimal schedule for this combination in clinical use. Received: 25 February 1997 / Accepted: 6 November 1997