Eleanor G. Zuhowski

Phase I Pharmacokinetic-Pharmacodynamic Study of 17-(Allylamino)-17-Demethoxygeldanamycin (17AAG, NSC 330507), a Novel Inhibitor of Heat Shock Protein 90, in Patients with Refractory Advanced Cancers

Purpose: 17-(Allylamino)-17-demethoxygeldanamycin (17AAG), a benzoquinone antibiotic, down-regulates oncoproteins by binding specifically to heat shock protein 90 (HSP90). We did a phase I study of 17AAG to establish the dose-limiting toxicity and maximum tolerated dose and to characterize 17AAG pharmacokinetics and pharmacodynamics. Experimental Design: Escalating doses of 17AAG were given i.v. over 1 or 2 hours on a weekly × 3 schedule every 4 weeks to cohorts of three to six patients. Plasma pharmacokinetics of 17AAG and 17-(amino)-17-demethoxygeldanamycin (17AG) were assessed by high-performance liquid chromatography. Expression of HSP70 and HSP90 in peripheral blood mononuclear cells was measured by Western blot. Results: Forty-five patients were enrolled to 11 dose levels between 10 and 395 mg/m2. The maximum tolerated dose was 295 mg/m2. Dose-limiting toxicity occurred in both patients (grade 3 pancreatitis and grade 3 fatigue) treated with 395 mg/m2. Common drug-related toxicities (grade 1 and 2) were fatigue, anorexia, diarrhea, nausea, and vomiting. Reversible elevations of liver enzymes occurred in 29.5% of patients. Hematologic toxicity was minimal. No objective responses were observed. 17AAG pharmacokinetics was linear. Peak plasma concentration and area under the curve of 17AG, the active major metabolite of 17AAG, increased with 17AAG dose, but the relationships were more variable than with 17AAG. 17AAG and 17AG in plasma were >90% protein bound. There were no consistent changes in peripheral blood mononuclear cell HSP90 or HSP70 content. Conclusions: 17AAG doses between 10 and 295 mg/m2 are well tolerated. 17AAG pharmacokinetics is linear. Peripheral blood mononuclear cell HSP90 and HSP70 are uninformative pharmacodynamic markers. The dose recommended for future studies is 295 mg/m2 weekly × 3, repeated every 4 weeks.

Cisplatin Preferentially Binds Mitochondrial DNA and Voltage-Dependent Anion Channel Protein in the Mitochondrial Membrane of Head and Neck Squamous Cell Carcinoma: Possible Role in Apoptosis

Purpose: Cisplatin adducts to nuclear DNA (nDNA) are felt to be the molecular lesions that trigger apoptosis, but the mechanism linking nDNA adduct formation and cell death is unclear. Some literature in the last decade has suggested a possible direct effect of cisplatin on mitochondria independent of nDNA interaction. In this study, we define separately the sequelae of cisplatin interactions with nDNA and with mitochondria in head and neck squamous cell carcinoma (HNSCC) cell lines. Experimental Design: Cisplatin binding to mitochondrial DNA (mtDNA) and proteins was analyzed by atomic absorption spectroscopy and other methods. Results: Following 1 hour of exposure to cisplatin, platinum adducts to mtDNA were 300- to 500-fold more abundant than adducts to nDNA; these differences were not due to differences in rates of adduct repair. Whereas HNSCC cell cytoplasts free of nDNA retained the same dose-dependent cisplatin sensitivity as parental cells, HNSCC ρ0 cells free of mtDNA were 4- to 5-fold more resistant to cisplatin than parental cells. Isolated mitochondria released cytochrome c within minutes of exposure to cisplatin, and ultrastructural analysis of intact HNSCC cells by electron microscopy showed marked mitochondrial disruption after 4 hours of cisplatin treatment, whereas the nucleus and other cellular structures remain intact. The very prompt release of cytochrome c from isolated mitochondria implies that apoptosis does not require alteration in mitochondrial gene transcription. Further, cisplatin binds preferentially to mitochondrial membrane proteins, particularly the voltage-dependent anion channel. Conclusions: Cisplatin binding to nDNA is not necessary for induction of apoptosis in HNSCC, which can result from direct action of cisplatin on mitochondria.

Suramin, an Active Drug for Prostate Cancer: Interim Observations in a Phase I Trial

BACKGROUND Previous studies indicate that suramin may be an active agent for treatment of solid tumors. The clinical use of suramin is complicated by a broad spectrum of toxic effects and complex pharmacology. Studies have suggested that the dose-limiting neurotoxicity of this agent is closely related to sustained plasma drug concentrations of 350 micrograms/mL or more. PURPOSE This phase I clinical trial in patients with solid tumors was designed to determine whether plasma concentrations resulting in both antitumor activity and manageable toxicity could be achieved with short, intermittent infusions of suramin. METHODS Thirty-seven patients, including 33 with metastatic, hormone-refractory prostate cancer, collectively received 43 courses of suramin designed to maintain a plasma concentration range of 200-300, 175-275, or 150-250 micrograms/mL. Patients received a test dose of 200 mg and an initial loading dose of 1000 mg/m2 on day 1 of therapy. Subsequent suramin doses and schedules were individually determined using a strategy of adaptive control with feedback, which used a maximum a posteriori Bayesian algorithm to estimate individual pharmacokinetic parameters. Patients were treated until dose-limiting toxicity or progressive disease developed. RESULTS Thirty-five of the 37 study patients and 31 of the 33 with prostate cancer were assessable for toxicity and response. Treatment was discontinued in 28 patients because of dose-limiting toxicity consisting of a syndrome of malaise, fatigue, and lethargy; recurrent reduction in creatinine clearance of 50% or more; or axonal neuropathy. Evidence of major antitumor activity was observed in patients with prostate cancer treated at all three plasma drug concentrations. Measurable responses (one complete response and five partial responses) were noted in six of 12 patients with measurable disease. Twenty-four (77%) of 31 patients had a reduction in prostate-specific antigen of 50% or more, and 17 (55%) of 31 had a reduction of 75% or more. Twenty (83%) of 24 patients reported reduction in pain. CONCLUSIONS Suramin can be safely administered as an intermittent bolus injection by use of adaptive control with feedback to control plasma drug concentrations; toxicity is significant but manageable and reversible. Suramin is active against hormone-refractory prostate cancer. IMPLICATIONS Future trials should address the role and necessary extent of therapeutic drug monitoring; the optimal plasma drug concentration range and duration of therapy; and the activity of suramin in combination with other agents, in earlier stages of prostate cancer, and in other tumor types.

Phase I Trial, Including Pharmacokinetic and Pharmacodynamic Correlations, of Combination Paclitaxel and Carboplatin in Patients With Metastatic Non–Small-Cell Lung Cancer

PURPOSE To determine the maximum-tolerated dose of paclitaxel with carboplatin with and without filgrastim support in patients with metastatic non-small-cell lung cancer (NSCLC) and to investigate the pharmacokinetics of paclitaxel and carboplatin and correlate these with the pharmacodynamic effects. PATIENTS AND METHODS Thirty-six chemotherapy-naive patients with metastatic NSCLC were entered into this phase I dose-escalation and pharmacokinetic study. Paclitaxel was initially administered as a 24-hour infusion at a fixed dose of 135 mg/m2, and the carboplatin dose was escalated in cohorts of three patients, using Calverts formula [dose(mg) = area under the concentration time curve (glomerular filtration rate + 25)], to target areas under the concentration time curve (AUCs) of 5, 7, 9, and 11 mg/mL x minute. A measured 24-hour urinary creatinine clearance was substituted for the glomerular filtration rate. Once the maximum-tolerated AUC (MTAUC) of carboplatin was reached, the paclitaxel dose was escalated to 175, 200, and 225 mg/m2. When the paclitaxel dose escalation began, the AUC of carboplatin was reduced to one level below the MTAUC. RESULTS Myelosuppression was the major dose-limiting toxicity. Thrombocytopenia was observed at a carboplatin AUC of 11 mg/mL x minute after course 2 and thereafter. End-of-infusion plasma paclitaxel concentrations and median duration of time above 0.05 microM were similar in course 1 versus course 2 at the 135 and 175 mg/m2 dose levels. The neutropenia experienced by patients was consistent with that observed in patients who had received paclitaxel alone. Measured carboplatin AUCs were approximately 12% (20% v 3% with course 1 v course 2, respectively) below the desired target, with a standard deviation of 34% at all dose levels. A sigmoid-maximum effect model describing the relationship between relative thrombocytopenia and measured free platinum exposure indicated that patients who received the combination of carboplatin with paclitaxel experienced less severe thrombocytopenia than would be expected from carboplatin alone. Of the 36 patients entered onto the study, one experienced a complete response and 17 had partial responses, for an overall response rate of 50%. The recommended doses of paclitaxel (24-hour infusion) and carboplatin for future phase II studies of this combination are (1) paclitaxel 135 mg/m2 with a carboplatin dose targeted to achieve an AUC of 7 mg/mL x minute without filgrastim support; (2) paclitaxel 135 mg/m2 with a carboplatin dose targeted to achieve an AUC of 9 mg/mL x minute with filgrastim support; and (3) paclitaxel 225 mg/m2 with a carboplatin dose targeted to achieve an AUC of 7 mg/mL x minute with filgrastim support. CONCLUSION The regimen of paclitaxel and carboplatin is well-tolerated and has promising activity in the treatment of NSCLC. There is no pharmacokinetic interaction between paclitaxel and carboplatin, but there is a pharmacodynamic, platelet-sparing effect on this dose-limiting toxicity of carboplatin.

Phase I and Pharmacodynamic Study of 17-(Allylamino)-17-Demethoxygeldanamycin in Adult Patients with Refractory Advanced Cancers

Purpose: The primary objective was to establish the dose-limiting toxicity (DLT) and recommended phase II dose of 17-(allylamino)-17-demethoxygeldanamycin (17AAG) given twice a week. Experimental Design: Escalating doses of 17AAG were given i.v. to cohorts of three to six patients. Dose levels for schedule A (twice weekly × 3 weeks, every 4 weeks) were 100, 125, 150, 175, and 200 mg/m2 and for schedule B (twice weekly × 2 weeks, every 3 weeks) were 150, 200, and 250 mg/m2. Peripheral blood mononuclear cells (PBMC) were collected for assessment of heat shock protein (HSP) 90 and HSP90 client proteins. Results: Forty-four patients were enrolled, 32 on schedule A and 12 on schedule B. On schedule A at 200 mg/m2, DLTs were seen in two of six patients (one grade 3 thrombocytopenia and one grade 3 abdominal pain). On schedule B, both patients treated at 250 mg/m2 developed DLT (grade 3 headache with nausea/vomiting). Grade 3/4 toxicities seen in >5% of patients were reversible elevations of liver enzymes (47%), nausea (9%), vomiting (9%), and headache (5%). No objective tumor responses were observed. The only consistent change in PBMC proteins monitored was a 0.8- to 30-fold increase in HSP70 concentrations, but these were not dose dependent. The increase in PBMC HSP70 persisted throughout the entire cycle of treatment but returned to baseline between last 17AAG dose of cycle 1 and first 17AAG dose of cycle 2. Conclusions: The recommended phase II doses of 17AAG are 175 to 200 mg/m2 when given twice a week and consistently cause elevations in PBMC HSP70.

Dominant-Negative Histone H3 Lysine 27 Mutant Derepresses Silenced Tumor Suppressor Genes and Reverses the Drug-Resistant Phenotype in Cancer Cells

Histone modifications and DNA methylation are epigenetic phenomena that play a critical role in many neoplastic processes, including silencing of tumor suppressor genes. One such histone modification, particularly at H3 and H4, is methylation at specific lysine (K) residues. Whereas histone methylation of H3-K9 has been linked to DNA methylation and aberrant gene silencing in cancer cells, no such studies of H3-K27 have been reported. Here, we generated a stable cell line overexpressing a dominant-negative point mutant, H3-K27R, to examine the role of that specific lysine in ovarian cancer. Expression of this construct resulted in loss of methylation at H3-K27, global reduction of DNA methylation, and increased expression of tumor suppressor genes. One of the affected genes, RASSF1, was shown to be a direct target of H3-K27 methylation-mediated silencing. By increasing DNA-platinum adduct formation, indicating increased access of the drug to target DNA sequences, removal of H3-K27 methylation resensitized drug-resistant ovarian cancer cells to the chemotherapeutic agent cisplatin. This increased platinum-DNA access was likely due to relaxation of condensed chromatin. Our results show that overexpression of mutant H3-K27 in mammalian cells represents a novel tool for studying epigenetic mechanisms and the Histone Code Hypothesis in human cancer. Such findings show the significance of H3-K27 methylation as a promising target for epigenetic-based cancer therapies.

Phase I Study of Paclitaxel in Combination With a Multidrug Resistance Modulator, PSC 833 (Valspodar), in Refractory Malignancies

PURPOSE To determine the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacokinetics of paclitaxel when given with PSC 833 (valspodar) to patients with refractory solid tumors. PATIENTS AND METHODS Patients were initially treated with paclitaxel 175 mg/m(2) continuous intravenous infusion (CIVI) over 3 hours. Subsequently, 29 hours of treatment with CIVI PSC 833 was started 2 hours before paclitaxel treatment was initiated. In this combination, the starting dose of paclitaxel was 52.5 mg/m(2). Paclitaxel doses were escalated by 17.5 mg/m(2) increments for four subsequent cohorts. Each cohort consisted of three patients with the exception of the last cohort, which consisted of six patients. Data for the pharmacokinetics of paclitaxel with and without concurrent PSC 833 administration were obtained. RESULTS All 18 patients completed at least one course of concurrent treatment (median, two courses; range, one to six) and were evaluable for toxicity. The MTD for paclitaxel with PSC 833 was 122.5 mg/m(2). Neutropenia was the DLT. All patients had PSC 833 blood concentrations greater than 1, 000 ng/mL before, during, and 24 hours after the paclitaxel infusion. PSC 833 produced small increases in the paclitaxel peak plasma concentrations and areas under the concentration-time curve. However, PSC 833 greatly prolonged the terminal phase of paclitaxel, resulting in plasma paclitaxel concentrations of more than 0.05 micromol/L for much longer than expected. As a result, myelosuppression was comparable to that produced by full-dose paclitaxel given without PSC 833. Of the 16 patients who were assessable for response, one patient experienced a partial response and an additional nine patients experienced disease stabilization after paclitaxel treatment alone. CONCLUSION Treatment with paclitaxel 122.5 mg/m(2) as a 3-hour CIVI concurrent with a 29-hour CIVI of PSC 833 results in acceptable toxicity. The addition of PSC 833 alters the pharmacokinetics of paclitaxel, which explains the enhanced neutropenia experienced by patients treated with this drug combination.

Phase I and pharmacokinetic trial of liposome-encapsulated doxorubicin

A total of 21 patients with advanced cancer were entered into a phase I study to determine the maximum tolerable dose (MTD) of liposome-encapsulated doxorubicin (LED) given weekly for 3 consecutive weeks at doses of 20, 30, or 37.5 mg/m2 per week. For a comparison of the pharmacokinetic behavior of LED with that of standard-formulation doxorubicin, 13 patients received a dose of standard-formulation doxorubicin 2 weeks prior to the first dose of LED. All doses were given by 1-h infusion through a central vein. Toxicity was evaluated in 22 courses delivered to 17 patients. The MTD with this schedule was 30 mg/m2 per week×3. The single patient treated at 37.5 mg/m2 weekly could not complete the entire course due to myelosuppression. At the dose of 30 mg/m2 per week, three of eight patients had grade ≥3 leukopenia. Other toxicities included mild to moderate thrombocytopenia, nausea, vomiting, fever, alopecia, diarrhea, fatigue, stomatitis, and infection. At the dose of 30 mg/m2 per week, the total doxorubicin AUC and peak total doxorubicin concentrations in plasma were 8.75±8.80 μM h (mean±SD) and 3.07±1.45 μM, respectively, after LED administration. The total doxorubicin AUC and peak total doxorubicin concentrations in plasma were 3.92±2.47 μM h and 2.75±2.70 μM, respectively, after the infusion of standard-formulation doxorubicin. The total body clearance of doxorubicin was 18.42±11.23 l/h after the infusion of LED and 31.21±15.48 l/h after the infusion of standard-formulation doxorubicin. The mean elimination half-lives of doxorubicin were similar: 8.65±5.16 h for LED and 7.46±5.16 h for standard-formulation doxorubicin. Interpatient variability in pharmacokinetic parameters as demonstrated by the percentage of coefficients of variation was 33%–105%. There was no relationship between the percentage of WBC decrease or the duration of WBC suppression and the total doxorubicin or doxorubicinol AUC. There was no correlation between the duration of leukopenia and drug exposure as reflected by the AUC of liposome-associated doxorubicin. LED can be given in doses similar to those of standard-formulation doxorubicin and produces acute toxicities similar to those caused by standard doxorubicin.

Plasma pharmacokinetics and tissue distribution in CD2F1 mice of Pc4 (NSC 676418), a silicone phthalocyanine photodynamic sensitizing agent.

Purpose: Pc4 is a silicone phthalocyanine photosensitizing agent that is entering clinical trials. Studies were undertaken in mice to develop a suitable formulation and analytical methodology for use in pharmacokinetic studies and to define the plasma pharmacokinetics, tissue distribution, and urinary excretion of Pc4 after i.v. delivery. Methods: An HPLC method suitable for separation and quantification of Pc4 was developed and validated for use in mouse plasma, tissues, and urine. The stability of Pc4 was characterized in a variety of formulations as well as in mouse plasma. Before pursuing pharmacokinetic studies, preliminary toxicity studies were undertaken. These studies utilized Pc4 formulated in diluent 12:0.154 M NaCl (1:3, v:v). Pharmacokinetic studies involved Pc4 doses of 40 mg/kg, 10 mg/kg and 2 mg/kg administered as i.v. boluses to female, CD2F1 mice . Doses of 40 mg/kg, 10 mg/kg, and 2 mg/kg were studied with drug formulated in diluent 12:0.154 M NaCl (1:3, v:v). Doses of 10 mg/kg and 2 mg/kg were also studied with drug formulated in a vehicle consisting of polyethylene glycol:Tween 80:0.01 M sodium phosphate buffer, pH 7.0 (40:0.2:59.8, v:v:v). Compartmental and non-compartmental analyses were applied to the plasma concentration-versus-time data. Concentrations of Pc4 were also determined in a variety of tissues, including brain, lung, liver, kidney, skeletal muscle, skin, heart, spleen, and abdominal fat. Urine was collected from animals treated with each of the doses of Pc4 mentioned above, and daily, as well as cumulative drug excretion was calculated until 168 h after treatment. Results: At a dose of 80 mg/kg, two of five male and two of five female mice were dead by 24 h after injection. Pathologic examination revealed gross findings of blue discoloration affecting many tissues, with lungs that were grossly hemorrhagic and very blue-black. Microscopic examination of the lungs revealed mild acute interstitial pneumonia, with perivascular edema and inflammation, and a detectable margination of neutrophils around larger pulmonary blood vessels. Animals sacrificed 14 days after treatment showed mild granulomatous pneumonia, characterized by clusters of multi-nucleated giant cells, with fewer macrophages and neutrophils. The giant cells frequently contained phagocytized particles, which were clear and relatively fusiform. All mice treated with 40 mg/kg or 20 mg/kg survived and returned to pretreatment weight during the 14 days after treatment. Intravenous bolus delivery of Pc4, at a dose of 40 mg/kg, produced “peak” plasma Pc4 concentrations between 7.81 and 8.92 μg/ml in mice killed at 5 min after injection (the earliest time studied after drug delivery). Sequential reduction of the Pc4 dose to 10 mg/kg in diluent 12:0.154 M NaCl (1:3, v:v), 10 mg/kg in polyethylene glycol:Tween 80:sodium phosphate buffer (40:0.2:59.8, v:v:v), 2 mg/kg in diluent 12:0.154 M NaCl (1:3, v:v), and, finally, 2 mg/kg in polyethylene glycol:Tween 80:sodium phosphate buffer (40:0.2:59.8, v:v:v) resulted in “peak” plasma Pc4 concentrations between 2.07 and 3.24, 0.68 and 0.98 μg/ml, and 0.29 and 0.41 μg/ml, respectively. Pc4 persisted in plasma for prolonged periods of time (72–168 h). Non-compartmental analysis of plasma Pc4 concentration-versus-time data showed an increase in area under the plasma Pc4 concentration-versus-time curve (AUC) when the dose of Pc4 increased from 2 mg/kg to 40 mg/kg. Across the 20-fold range of doses studied, total body clearance (CLtb) varied from 376 to 1106 ml h−1 kg−1. Compartmental modeling of plasma Pc4 concentration versus time data showed the data to be fit best by a two-compartment, open, linear model. Minimal amounts of Pc4 were detected in the urine of mice. After i.v. bolus delivery to mice, Pc4 distributed rapidly to all tissues and persisted in most tissues for the duration of each pharmacokinetic study. Tissue exposure, as measured by AUC, increased in a dose-dependent fashion. Conclusions: The HPLC method developed for quantification of Pc4 in plasma, urine, and tissues should be suitable for clinical studies of the drug. Pc4 is widely distributed and persists in plasma and tissues of mice for prolonged periods of time. These data are relevant to the design of forthcoming clinical trials of Pc4.

Evaluation of Plasma Insulin-like Growth Factor Binding Protein 2 and Her-2 Extracellular Domain as Biomarkers for 17-Allylamino-17-Demethoxygeldanamycin Treatment of Adult Patients with Advanced Solid Tumors

Purpose: Interaction of 17-allylamino-17-demethoxygeldanamycin (17-AAG) with heat shock protein 90 results in proteasomal degradation of many proteins, including Her-2-neu, with subsequent decreased expression of insulin-like growth factor binding protein-2 (IGFBP-2). Concentrations of both IGFBP-2 and Her-2 extracellular domain (Her-2 ECD) in sera of mice bearing BT474 human breast cancer xenografts decrease after 17-AAG treatment. We investigated whether this phenomenon occurred in patients. Materials and Methods: Eight to 15 plasma samples were obtained between 0 and 72 h from 27 patients treated with single-agent 17-AAG at doses between 10 and 307 mg/m2 and 18 patients treated with 17-AAG at doses between 220 and 450 mg/m2 combined with 70 to 75 mg/m2 of docetaxel. Pretreatment plasma samples were also obtained from 12 healthy volunteers. Plasma IGFBP-2 and Her-2 ECD concentrations were quantitated by ELISA. Results: Pretreatment plasma IGFBP-2 concentrations in patients (171 ± 116 ng/mL) were 2-fold higher than those in healthy volunteers (85 ± 44 ng/mL; P < 0.05). Following 17-AAG treatment, there were no consistent dose-dependent or time-dependent changes in plasma IGFBP-2 and Her-2 ECD concentrations. IGFBP-2 concentrations decreased by ≥40% in 8 patients, increased 2- to 5-fold in 8 patients, and remained essentially unchanged in 29 patients. Her-2 ECD concentrations decreased by ≥40% in 10 patients, increased 1.5- to 5-fold in 2 patients, and remained essentially unchanged in 25 patients. Conclusions: As previously reported, IGFBP-2 concentrations in plasma of cancer patients are significantly higher than those in healthy volunteers. In contrast to a mouse model, 17-AAG treatment was not consistently associated with decreases in IGFBP-2 or Her-2 ECD concentrations in patient plasma.