Proteasome inhibition with bortezomib (PS-341): a phase I study with pharmacodynamic end points using a day 1 and day 4 schedule in a 14-day cycle.

PURPOSE: We performed a phase I study of a day (D) 1 and D4 bortezomib administration once every 2 weeks to determine the recommended phase II dose and toxicity profile, and the extent of 20S proteasome inhibition obtained. PATIENTS AND METHODS: Patients with solid tumors or lymphomas were treated with bortezomib at 0.25 to 1.9 mg/m2 on D1 and D4, every 2 weeks. 20S proteasome levels in blood were assayed at baseline and at 1, 4, and 24 hours postdose in cycle 1. RESULTS: On this D1 and D4 every 2 weeks' schedule, dose-limiting toxicity (DLT) was evident at the 1.75 and 1.9 mg/m2 dose levels, most commonly in patients receiving individual total doses > or = 3.0 mg. The main DLT was peripheral neuropathy evident at the higher doses and in patients previously exposed to neurotoxic agents. Other DLTs included diarrhea and fatigue; grade 3 thrombocytopenia was also noted. Reversible inhibition of 20S proteasome activity was dose dependent and best fit a total dose (mg) per fraction rather than mg/m2; 70% of baseline activity was inhibited by a dose of 3.0 to 3.5 mg given on D1 and on D4 every other week. Antitumor effects short of confirmed partial responses were observed in patients with melanoma, non-small-cell lung cancer, and renal cell carcinoma. CONCLUSION: Bortezomib (PS-341) is a novel antineoplastic agent that is well tolerated at doses not exceeding 3.0 mg (equivalent to 1.75 mg/m2), repeated on D1 and D4 every other week. This dose correlates with 70% inhibition of 20S proteasome activity. DLTs include neuropathy, fatigue, and diarrhea.

Modulation of monocyte functions by muramyl tripeptide phosphatidylethanolamine in a phase II study in patients with metastatic melanoma.

BACKGROUND: Muramyl tripeptide phosphatidylethanolamine (MTP-PE) is a synthetic analogue of muramyl dipeptide (MDP), a component of bacterial cell walls that has potent in vitro monocyte-activating properties. We conducted a phase II clinical trial of MTP-PE in 30 patients with metastatic melanoma. PURPOSE: Our purpose was to define a clinical response rate for this agent in patients with advanced melanoma and to evaluate the agent's immunomodulatory properties. METHODS: Patients were randomly assigned to 1- or 4-mg dose levels of MTP-PE and received the drug intravenously once a week for 12-24 weeks. Immunological monitoring consisted of measurement of plasma tumor necrosis factor-alpha (TNF-alpha), neopterin, interleukin-1-beta, interleukin-6 (IL-6), and beta 2-microglobulin levels; phenotyping analysis of expression of human HLA-DR, CD-14 on mononuclear cells; and measurement of in vitro monocyte cytotoxicity against SKMel28 targets cells. RESULTS: MTP-PE was well tolerated; fever and chills were the major toxic effects. Plasma TNF-alpha levels increased 16-fold 2 hours after the first MTP-PE treatment. Increases in TNF-alpha levels after MTP-PE administration continued through week 12, but changes were of a lower magnitude after week 1. Plasma neopterin levels were significantly increased 24 hours after treatment at weeks 1, 6, and 12. A marked increase in IL-6 and a modest rise in beta 2-microglobulin levels were also seen at week 1. No significant changes from baseline IL-1 beta were observed. In the cytotoxicity assay, monocyte cytotoxic activity was significantly increased at weeks 4 and 6. Surface immuno-phenotyping revealed a consistent transient reduction in the number of circulating monocytes 2 hours after MTP-PE was administered. In addition, we observed a down-regulation (i.e., a decrease) in the expression of Leu M3 and HLA-DR on monocytes, 2 hours after MTP-PE treatment, followed by a recovery 24 hours after treatment. No objective clinical responses were seen in this advanced disease population. CONCLUSIONS: We conclude that MTP-PE has pleiotropic and potentially beneficial biologic effects and that further clinical investigations of MTP-PE are justified. IMPLICATIONS: In view of the clear immunomodulatory actions seen in our study and in earlier clinical trials, we believe that MTP-PE deserves further study in the adjuvant setting.

Pharmacokinetics of the cardioprotector ADR-529 (ICRF-187) in escalating doses combined with fixed-dose doxorubicin.

BACKGROUND: Although doxorubicin is an anticancer agent with a wide spectrum of activity, therapy with this anthracycline must often be discontinued at a time of benefit to the patient because of the drug's cumulative cardiotoxicity. ICRF-187 (ADR-529, dexrazoxane) is a bisdioxopiperazine compound that protects against cardiac toxicity induced by doxorubicin. PURPOSE: Our objectives in this study were to determine the maximum tolerated dose of ADR-529 (which uses a different vehicle than ICRF-187) when given with a fixed doxorubicin dose and to determine whether ADR-529 alters doxorubicin pharmacokinetics. METHODS: Twenty-five patients were treated with doxorubicin (60 mg/m2) preceded by administration of ADR-529 in escalating dosages (i.e., 60, 300, 600, 750, and 900 mg/m2) to groups of three to nine patients. ADR-529 was administered over a 15-minute period beginning 30 minutes before doxorubicin treatment; the protocol was repeated every 3 weeks. Blood was sampled frequently for drug levels, which were determined by high-pressure liquid chromatography with fluorescence (doxorubicin) and electrochemical detection (ADR-529). RESULTS: Dose-limiting neutropenia occurred in four of six previously treated patients at an ADR-529 dose of 600 mg/m2; the dose ratio of ADR-529 to doxorubicin was 10:1. For three additional patients with better Eastern Cooperative Oncology Group performance status and a maximum of one prior chemotherapy regimen, 600 mg/m2 was tolerated, but grade 3 or 4 neutropenia occurred in four of six patients who received an ADR-529 dose of 900 mg/m2 and in three of four patients at a dose of 750 mg/m2. Doxorubicin's estimated terminal half-life was 39.5 +/- 18.3 (mean +/- SD) hours; the area under the curve for plasma concentration of drug x time (AUC) was 1.74 +/- 0.40 (micrograms/microL) x hour. Total-body clearance was 598 +/- 142 microL/m2 per minute (N = 20), and it did not vary with ADR-529 dose. Estimated distribution and elimination phase half-lives for plasma ADR-529 were 0.46 +/- 0.30 hours and 4.16 +/- 2.94 hours, respectively. Total-body clearance was 111 +/- 87 microL/m2 per minute (N = 18); AUC was linear (r2 = .92), and the clearance rate was constant (r2 = .18) from 60 to 900 mg/m2. CONCLUSIONS: Myelotoxicity was dose limiting for ADR-529 at 600-750 mg/m2 when given with a fixed dose of doxorubicin at 60 mg/m2 (dose ratios of ADR-529 to doxorubicin ranged from 10:1 to 12.5:1). When used in combination, ADR-529 did not perturb doxorubicin's distribution, metabolism, or excretion; therefore, other mechanisms of cardioprotection must be involved. IMPLICATIONS: We recommend that an ADR-529 dose of 600 mg/m2 be given with single-agent doxorubicin at a dose of 60 mg/m2 in future studies.

Comparison of lipid composition and 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization measurements of hairy cells with monocytes and lymphocytes from normal subjects and patients with chronic lymphocytic leukemia.

In this report, we compare the lipid composition and fluorescence polarization properties of hairy cells with those of monocytes and lymphocytes from normal subjects and of lymphocytes from patients with chronic lymphocytic leukemia. For hairy cells, the cholesterol content was 4.66 +/- 1.49 (S.D.) mumol/10(9) cells, and the cholesterol/phospholipid ratio was 0.60 +/- 0.09. These were significantly higher than the values of normal lymphocytes, (cholesterol content, 2.75 +/- 0.65 mumol; cholesterol/phospholipid ratio, 0.50 +/- 0.07) or of chronic lymphocytic leukemia lymphocytes (cholesterol content, 1.76 +/- 0.43 mumol; cholesterol/phospholipid ratio, 0.44 +/- 0.07). Normal monocyte values (cholesterol content, 5.81 +/- 2.08 mumol; cholesterol/phospholipid ratio, 0.59 +/- 0.06) were similar to those of hairy cells. Using the probe 1,6-diphenyl-1,3,5-hexatriene, the fluorescence polarization value at 25 degrees for hairy cells was 0.302, compared to the value of 0.259 obtained with chronic lymphocytic leukemia lymphocytes. Intermediate values (0.294) were obtained with normal lymphocytes and monocytes. Fluorescence polarization values were higher in hairy cell membranes than in chronic lymphocytic leukemia lymphocyte membranes, indicating a low fluidity in the former cell, compatible with their higher cholesterol content and cholesterol/phospholipid ratio. These studies show that two neoplastic cells, hairy cells and chronic lymphocytic leukemia lymphocytes, differ markedly in membrane fluidity and that a high membrane fluidity does not necessarily occur in neoplasia.

Chlorambucil pharmacokinetics and DNA binding in chronic lymphocytic leukemia lymphocytes.

Chlorambucil (CLB) uptake by chronic lymphocytic leukemia lymphocytes was studied using a radiometric and a newly developed high-performance liquid chromatography assay. CLB labeled with 14C in either the chloroethyl group or phenyl ring was used with identical results. Drug accumulation by the cells was found to peak at 30 s, was independent of temperature, and was proportional to medium CLB concentration over a wide range. Efflux from cells loaded with CLB and resuspended in drug-free medium was nearly complete at 30 s. The metabolic inhibitors 2-deoxyglucose and NaN3, the nitrogen mustard transport inhibitor hemicholinium-3, and another alkylating agent, melphalan, had no effect on drug uptake. We conclude that CLB enters and exits chronic lymphocytic leukemia lymphocytes by simple diffusion. Cells from 17 patients with all stages of chronic lymphocytic leukemia were studied including three with CLB-resistant disease, and no heterogeneity was found in the peak cell-associated CLB content or in metabolite pattern on high-performance liquid chromatography. These findings make it unlikely that transport or cellular drug metabolism are factors in drug resistance. Drug-DNA binding was found to be temperature-sensitive and increased with time of incubation. Gel filtration of DNA before and after enzymatic digestion indicated the presence of drug-DNA adducts. High-performance liquid chromatography analysis of digested DNA and DNA treated by neutral thermal hydrolysis suggested the presence of multiple adducts. Most of the radioactivity was found as purine adducts. Studies with CLB labeled at two different sites revealed the presence of the phenyl group and ethyl chains in the adducts. A survey of patients showed increased drug-DNA binding in cells from patients with clinical CLB resistance.

Relationship of dehydroascorbic acid transport to cell lineage in lymphocytes from normal subjects and patients with chronic lymphocytic leukemia.

Dehydroascorbic acid is the principal form for the cellular uptake by blood cells of vitamin C. Since previous studies from this laboratory had shown a higher content of ascorbic acid and dehydroascorbic acid (DHA) in chronic lymphocytic leukemia (CLL) lymphocytes when compared to their normal counterparts, DHA uptake was characterized using these cells. The affinities of CLL and normal lymphocytes for DHA uptake were similar, as demonstrated by the Km values of 3.7 and 3.5 mM, respectively. Differences were found in other kinetic constants of DHA uptake. The Vmax for normal lymphocytes, 634 mumol/liter cell H2O/min, was approximately twice that of CLL cells, 392 mumol/liter cell H2O/min. In addition, the initial velocity and the maximal DHA uptake by normal lymphocytes were greater than that of CLL lymphocytes. These differences were not simply a reflection of lymphocyte subsets since CLL B-cells demonstrated lower uptake rates than did normal B-cells whereas CLL T-cells were similar to their normal counterparts. The alterations appear to be specific for the leukemic B-cell since they were not shared by neoplastic cells from two patients with T-cell CLL. When analyzed in light of the 3-fold greater cellular DHA and ascorbic acid content in B-cell CLL as compared to normal lymphocytes, these kinetic parameters support the occurrence of a concentration-dependent transport system for DHA. We conclude that the DHA uptake properties of CLL lymphocytes of B-cell origin serves to distinguish this lineage from T-cell CLL or normal lymphocytes.

Phase I trial of cyclophosphamide, doxorubicin, and 5-fluorouracil plus interferon-alpha 2b in patients with advanced breast cancer.

alpha-Interferon (IFN-alpha) enhances the activity of 5-fluorouracil in patients with advanced colorectal carcinoma. Preclinical evidence suggests a similar potential role for IFN-alpha combined with cyclophosphamide, doxorubicin (Adriamycin, Adria Laboratories, Columbus, OH), and 5-fluorouracil (CAF) in advanced adenocarcinoma of the breast. To determine a maximum tolerated dose of IFN-alpha that could be combined with CAF and that did not compromise CAF dose intensity and to determine the effect of IFN-alpha on the pharmacokinetics of doxorubicin, a phase I study of IFN-alpha plus CAF was performed by the Eastern Cooperative Oncology Group. Nine patients with advanced breast cancer received CAF (cyclophosphamide at 100 mg/m2/day p.o. on days 1-14, doxorubicin at 30 mg/m2 and 5-fluorouracil at 500 mg/m2 i.v. bolus on days 1 and 8) plus IFN-alpha (1 milliunit/m2, n = 6, or 2 milliunits/m2, n = 3) given s.c. on days 1, 3, 5, and 8 (1 h prior to the doxorubicin and 5-FU injection on days 1 and 8) of each cycle every 28 or more days. Escalation of the IFN-alpha dose occurred in cohorts of 3-6 patients if a dose-limiting toxic event (neutropenic fever, platelet nadir of < 25,000/microliters, > 2-week treatment delay, or a > 50% dose reduction in day 8 CAF) occurred during the first two cycles in 0 of 3 or 1 of 6 patients. During cycle 1, IFN-alpha was omitted on day 1, and multiple plasma samples were drawn on day 1 (without IFN-alpha) and day 8 (with IFN-alpha) after each doxorubicin injection and were analyzed for plasma doxorubicin concentration. The maximum tolerated dose of IFN-alpha by our criteria was 1 milliunit/m2, and neutropenia was the predominant toxic effect that precluded IFN-alpha dose escalation. The dose intensity of CAF achieved with IFN-alpha was identical to that for CAF alone observed in prior studies. IFN-alpha had no significant effect on the pharmacokinetics of doxorubicin, although 3 of 7 patients studied had reduced doxorubicin clearance, ranging from 32% to 69%. Alternative CAF drug delivery schedules (all drugs given i.v. every 3-4 weeks) that are more amendable to hematopoietic growth factor support may be more suitable to combine with higher doses of IFN-alpha that may produce modulation.

Ribonucleotide content of mononuclear cells from normal subjects and patients with chronic lymphocytic leukemia: increased nicotinamide adenine dinucleotide concentration in chronic lymphocytic leukemia lymphocytes.

The ribonucleotide content of lymphocytes obtained from normal subjects and patients with chronic lymphocytic leukemia (CLL) was determined by means of high-performance liquid chromatography. The levels of normal B- and T-cells were compared to each other as well as those of their CLL counterparts. Unfractionated CLL lymphocytes, predominantly B-cells, had significantly lower levels of adenosine-5'-triphosphate, cytidine-5'-triphosphate, uridine-5'-triphosphate, cytidine-5'-diphosphate, and guanosine-5'-phosphate, while the concentration of nicotinamide-adenine dinucleotide was significantly higher than in normal unfractionated lymphocytes which consisted mainly of T-cells. For enriched populations: (a) CLL B-cells had much lower adenosine-5'-triphosphate (3439 versus 5689) (pmol/1 X 10(7) cells), cytidine-5'-triphosphate (107 versus 313), guanosine-5'-triphosphate (462 versus 978), and uridine-5'-triphosphate (633 versus 1214) than normal B-cells; (b) CLL T-enriched subpopulations had significantly lower ribonucleoside triphosphates, adenosine-5'-triphosphate (3217 versus 5468), cytidine-5'-triphosphate (119 versus 209), guanosine-5'-triphosphate (422 versus 826), and uridine-5'-triphosphate (504 versus 969) than normal T-cells. The lower ribonucleoside triphosphate levels found in unfractionated CLL lymphocytes, therefore, are the result of differences between the CLL and normal B-cells as well as between CLL and normal T-cells. These findings establish a framework for studying the reasons underlying the decreased ribonucleoside triphosphate levels in unfractionated CLL lymphocytes. T-helper and T-suppressor lymphocytes showed similar ribonucleotide patterns. Nucleoside and base levels were significantly higher in normal monocytes than in normal lymphocytes. The only compound found to be increased in the CLL B-lymphocytes when compared to their normal counterparts was nicotinamide-adenine dinucleotide. The level in CLL lymphocytes was 404 versus 209 pmol/10(7) cells for normal B-lymphocytes. No correlation was found between any ribonucleotide levels and the expression of 5'-nucleotidase activity.

Purification and characterization of actin from normal and chronic lymphocytic leukemia lymphocytes.

Previous studies from this laboratory have shown actin to be a major protein of human lymphocytes (Stark, R., Liebes, L. F., Nevrla, D., and Silber, R. Biochem. Med., 27: 200-206, 1982). We now report the purification to homogeneity and characterization of actin from blood lymphocytes of normal subjects and patients with chronic lymphocytic leukemia. The recovery of the purified protein was about 20%. The properties of the lymphocyte actins were compared to each other and to those of rabbit skeletal muscle actin. Lymphocyte actin consisted of beta and gamma forms in a 2:1 ratio. The Mr 42,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Normal and leukemic lymphocyte actin had similar polymerization properties as assessed by viscosity measurements at 25 degrees and 4 degrees, and the ultrastructural appearance of the filaments was the same. Similar patterns were observed between normal and chronic lymphocytic leukemia actin tryptic digests analyzed by high-performance liquid chromatography. The Vmax of the actin-activated myosin Mg2+ ATPase activity was compared using rabbit skeletal muscle heavy meromyosin and subfragment 1 preparations. The values obtained with rabbit skeletal muscle and normal lymphocyte actin were identical. The Vmax observed with chronic lymphocytic leukemia lymphocyte actin was 70% of that obtained with normal lymphocyte actin. The amount of actin needed to produce half-maximal activation (Kapparent) of heavy meromyosin and subfragment 1 were, respectively, 26 and 25 microM for normal lymphocytes and 18 and 24 microM for chronic lymphocytic leukemia lymphocytes. The anomalous ATP activation by actin did not reflect differences in B-:T-cell subpopulations between chronic lymphocytic leukemia and normal lymphocytes. The possible significance of the observed differences between the myosin Mg2+ ATPase activation by chronic lymphocytic leukemia and normal lymphocyte actin is discussed.

Radiosensitization of tumor-targeted radioimmunotherapy with prolonged topotecan infusion in human breast cancer xenografts.

Clinical radioimmunotherapy (RIT) of solid tumors holds great promise, but as yet has been unable to deliver tumoricidal radiation doses without unacceptable toxicity. Our experimental approach aims to potentiate the therapeutic action of radioimmunoconjugates at the tumor site and thus improve the efficacy of RIT by combination with other treatment modalities. The topoisomerase I inhibitors are a unique class of chemotherapeutic agents that interfere with DNA breakage-reunion by inhibiting the action of topoisomerase I. Preclinical studies suggest that prolonged infusion of topoisomerase I inhibitors enhances cell toxicity due to ionizing radiation. We evaluated the efficacy of combined treatment with continuous administration of topotecan and 90Y-MX-DPTA BrE3 monoclonal antibody (which recognizes an epitope of breast epithelial mucin expressed in most breast cancers) on human mammary carcinoma xenografts in nude mice. Topotecan or 90Y-BrE3 treatment alone delayed overall tumor growth rate transiently but did not affect survival. The combination of RIT with topotecan substantially reduced growth of relatively large established tumors and caused complete tumor regressions and prolonged tumor-free survival in a substantial proportion of treated animals. In vitro studies demonstrated an increase in apoptotic rate and a decrease in cell proliferation of tumor cell lines treated with this combination. We combined the radiosensitization property of topotecan and the specificity of systemic RIT to establish a novel therapy for solid tumors in an experimental tumor xenograft model.

Phase I trial of escalating dose doxorubicin administered concurrently with alpha 2-interferon.

The clinical use of alpha 2-interferon and doxorubicin is based on in vitro and preclinical in vivo observations of synergistic antitumor efficacy. To test this combination a Phase I clinical and pharmacokinetic study of the concurrent use of alpha 2-interferon and doxorubicin was initiated in patients with malignant solid tumors. Each 5-wk treatment cycle consisted of 3 wk of drug administration and 2 wk of rest. The alpha 2-interferon was administered s.c. at a constant dose of 10 million IU/m2 on Mondays, Wednesdays, and Fridays in all patients while the doxorubicin was administered weekly beginning with a dose of 5 mg/m2 and escalated to the maximum tolerated dose of 25 mg/m2. At least three evaluable patients were entered at each dose level, and no dose escalations were allowed within patients. The dose-limiting toxicities were granulocytopenia and thrombocytopenia. Hepatic enzyme elevations and systemic symptoms due to interferon occurred at all dose levels. None was severe or dose limiting, and all were reversible. These toxicity data suggest that the hepatotoxic effects of interferon do not enhance doxorubicin toxicity when given by this dose and schedule. Doxorubicin plasma levels were measured at each dose level. The recommended dose of doxorubicin is 25 mg/m2 per wk when administered with 10 million IU/m2 of interferon in this schedule. This schedule allows for the administration of a greater total dose of doxorubicin than has been achieved when given every 3 wk with the same dose and schedule of alpha 2-interferon in a parallel study.

The solution behavior of the bovine myelin basic protein in the presence of anionic ligands. Binding behavior with the red component of trypan blue and sodium dodecyl sulfate.

The interaction of the azo dye (2,3'-dimethyldiphenyl-7-azo-8-amino-1-napthol 3,6-disulfonic acid (TBR) and sodium dodecyl sulfate with the bovine myelin basic protein has been studied using absorbance, circular dichroism and 220 MHz PMR spectroscopy. Additional analyses of the binding reaction were carried out using light scattering, ultracentrifugal and electrophoretic techniques. A procedure for preparing pure TBR was developed. A modified structure for this synthesized TBR has been suggested. The mechanism of TBR binding to the myelin basic protein was found to be metachromatic. In addition, the interaction of TBR with the basic protein which gives rise to aggregation of the dye bound species was found to be analogous to the model proposed by Schwarz, G. and Seelig-Löffler, A. ((1975) Biochim. Biophys. Acta 379, 125-138) to explain the binding of acridine orange with poly (alpha-L-glutamic acid). PMR spectral analyses suggested that arginine residues provide the majority of primary sites of attachment on the basic protein for TBR. The effect of sodium dodecyl sulfate binding with the bovine myelin basic protein was found to induce a minimal change in the conformation of the protein. The induction of only about 20% alpha helial structure could be demonstrated and the binding was reversed by raising the solution temperature to 73 degrees C. The difference in the observed behavior of basic protein arising from TBR binding as opposed to the binding of sodium dodecyl sulfate is viewed as resulting from two different binding mechanisms. The binding behavior of TBR is primarily a consequence of charge-charge interaction while the binding effects of sodium dodecyl sulfate are a consequence of hydrophobic interaction. The sodium dodecyl sulfate binding acts as a shield which limits charge-charge interaction in the basic protein molecule thus preventing aggregate formation while TBR imposes no such restraints.

Solution behavior, circular dichroism and 22 HMz PMR studies of the bovine myelin basic protein.

Bovine myelin basic protein has been investigated with regard to its solution behavior, circular dichroism and 220 MHz PMR spectral properties. At pH 4.8 gamma/2=0.1 acetate buffer, light scattering yielded a Mr of 17 700 and a virial coefficient of 1.0-10(-4) mol-ml/g2. Above pH 7.0 the protein was found to aggregate to higher mol. wt species. Sedimentation experiments at pH 4.8 yielded s degrees 20,w of 1.27 S at gamma/2=0.1 and 1.46 S at gamma/2=0.35. The diffusion coefficient determined from ultracentrifugal experiments was 7.25-10(-7) cm2/s at gamma/2=0.1 and 0.35. The value of f/f0 from diffusion at pH 4.8 and gamma/2=0.35 was 1.64, corresponding to an axial ratio of 11 to 1. The radius of gyration was calculated as 4.28 nm and the root mean square end to end distance was 10.5 nm. At pH 9.0, gamma/2=0.1, s degrees 20,w was 1.71 S and D degrees 20,w was estimated at 7.4-10(-7) cm2/s. The behavior at pH 9.0 reverted to the behavior at pH 4.8 when the pH was readjusted. The E1%/1cm=5.64 at 276.4 nm and 225 at 196 nm. Titration of the protein with trifluoroethanol elicited three distinct regions of conformation stability having increasing helical content as the mol fraction of trifluoroethanol increased. The results of the present study have permitted some comparison of analogous properties and conformational behavior with the basic membrane protein cytochrome c.

A reappraisal of leukocyte dehydroascorbate reductase.

Dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1) activity was determined in human leukocyte homogenates using a direct spectrophotometric assay. Despite previous studies, using a less sensitive coupled assay, which reported that this enzyme was present in leukocytes, we found that neither neutrophil nor chronic lymphocytic leukemia lymphocyte extracts had detectable activity. Furthermore, when the product was quantitated by HPLC, protein-dependent generation could not be demonstrated. Mixing experiments with a partially purified enzyme preparation from spinach leaves provided no evidence for the presence of an inhibitor in neutrophil homogenates. These findings suggest that in human leukocytes, dehydroascorbate reduction does not occur enzymatically.

Human lymphocyte tubulin. Purification and characterization in normal and leukemic cells.

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119000. The protein was shown to consist of two subunits, with molecular weights of 61000 and 58000 comparable to the alpha and beta polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 +/- 0.86 . 10(6) M-1 and a level in normal lymphocytes of 1.21 . 10(2) +/- 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and Ka for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocytes tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity for function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.

Fluorescent labeling of DNA in solution with covalently bound acriflavin.

We have prepared a fluorescent derivative of DNA based on the acriflavin-Feulgen histological procedure for staining DNA. Our procedure involved binding acriflavin to DNA in solution by reacting the acriflavin with aldehydes formed on the deoxyribose of DNA by controlled removal of a few percent of the purine bases of the DNA. Partially depurinated DNA was reacted with the acriflavin reagent, and unbound acriflavin was removed by chromatography on Sephadex G-25 eluted with phosphate buffered guanidine -HCl. Such single-stranded depurinated DNA bound 0.36 acriflavin molecules per 100 purine bases per h of depurination. DNA containing one bound acriflavin per 200 bases reassociated at 85% of the value of control DNA. The acriflavin - DNA complex showed new absorption maxima at 466 and 370 nm. The fluorescent product had excitation maxima at 304 and 465 nm and an emission maximum at 502 nm. This labeling procedure should be useful in place of or in addition to radioactive labeling for DNA.

Phase I trial of low-dose continuous topotecan infusion in patients with cancer: an active and well-tolerated regimen.

PURPOSE: The objective of this trial was to define the maximum-tolerated dose (MTD) of topotecan for a 21-day infusion schedule, repeated every 28 days, in patients with cancer. PATIENTS AND METHODS: Cohorts of four patients received continuous ambulatory infusions of topotecan in escalated duration with doses beginning at 0.20 mg/m2/d for 7 days. Forty-four patients with a histologic diagnosis of cancer refractory to standard therapy were treated with infusions of topotecan for a total of 115 cycles and 1,780 patient-days of infusion. The median number of treatment cycles per patient was two (range, one to eight). All patients were heavily pretreated with chemotherapy and/or radiation. RESULTS: The dose-limiting toxicity (DLT) was myelo-suppression, with thrombocytopenia greater than neutropenia seen at the dose level of 0.70 mg/m2/d for 21 days. At the MTD of 0.53 mg/m2, ten patients were treated for a total of 20 courses, resulting in one episode of grade 4 thrombocytopenia and leukopenia, one grade 3 thrombocytopenia, and two grade 3 leukopenias. This dose regimen was well tolerated, with minimal nonhematologic toxicity. Local infusion port complications developed in two patients and two had bacteremia, including one patient with repeated local skin infections. Objective responses were observed in this heavily pretreated population for patients with ovarian cancer (two partial responses and one mixed response in six patients), breast cancer (one partial response and one mixed response in two patients), and for one patient each with renal and non-small-cell lung cancer (two partial remissions). CONCLUSION: Twenty-one-day topotecan infusion is well tolerated at 0.53 mg/m2, with dose-intensity exceeding other schedules for administration of topotecan. The DLT is hematologic, with thrombocytopenia somewhat exceeding leukopenia. Objective responses were observed in seven patients with breast, ovarian, renal, and non-small-cell lung cancer.

Activity and pharmacodynamics of 21-Day topotecan infusion in patients with ovarian cancer previously treated with platinum-based chemotherapy. New York Gynecologic Oncology Group.

PURPOSE: Twenty-one-day topotecan infusion was administered as second-line therapy in patients with previously treated ovarian cancer (based on our prior favorable phase I experience) to determine its activity, time to progression, and pharmacodynamics. PATIENTS AND METHODS: Ovarian cancer patients with measurable lesions and one prior platinum-containing regimen were eligible. Topotecan 0.4 mg/m(2)/d 21-day continuous ambulatory intravenous infusion, with appropriate dose modifications for toxicity, was administered every 28 days. Weekly blood levels of topotecan and topoisomerase-1 (topo-1) levels in peripheral-blood mononuclear cells (PBMCs) were determined for pharmacodynamic correlation. RESULTS: Twenty-four patients were entered onto the study (six cisplatin-refractory, five relapsing within < 6 months and 13 relapsing > 6 months after platinum-based therapy). A total of 128 cycles of topotecan (median, four cycles per patient; range, one to 12 cycles) were administered. The major toxicity was neutropenia (29% grade 3 in all cycles and 4% grade 4). One episode of grade 4 thrombocytopenia (4%) occurred. Fifty-two percent of the patients had anemia that required transfusions. Eight of 23 patients with measurable disease (35%; 95% confidence interval [CI], 15% to 54%) had partial responses (PRs) lasting longer than 1 month. Two of these patients had minor residual computed tomographic changes but had clinical complete remissions that lasted up to 53 weeks while they were not undergoing further therapy. One patient with nonmeasurable disease had a PR (by CA-125 criteria) that lasted 6 months, for an overall response rate of 38% in nine of 24 patients (95% CI, 18% to 57%). The median time to progression was 26 weeks. Pharmacodynamic analysis demonstrated a statistically significant decrease in free PBMC topo-1 level at weeks 2 and 3 of drug administration. There was a strong statistical correlation between the decrease in free topo-1 levels and increasing area under the curve (AUC) for topotecan. This was confirmed in a pharmacodynamic model. CONCLUSION: Twenty-one-day infusion is a well-tolerated method of administering topotecan. Pharmacodynamic studies demonstrate correlations between (1) the week of infusion and the PBMC topo-1 level, (2) the AUC of topotecan and the decrease in topo-1 levels, and (3) the change in topo-1 level and the neutrophil nadir. The objective response rate of 35% to 38% (95% CI, 15% to 57%) in this small multicenter study is at the upper level for topotecan therapy in previously treated ovarian cancer. Prolonged topotecan administration therefore warrants further investigation in larger, randomized studies comparing this 21-day schedule with the once-daily-for-5-days schedule.

Phase I trial of escalating doses of paclitaxel plus doxorubicin and dexrazoxane in patients with advanced breast cancer.

PURPOSE: To determine the maximum-tolerable dose (MTD) of paclitaxel given as a 3-hour intravenous (IV) infusion that could be used in conjunction with doxorubicin and dexrazoxane, and to determine the effect of dexrazoxane on the pharmacokinetics of paclitaxel and doxorubicin. PATIENTS AND METHODS: Twenty-five patients with advanced breast cancer received dexrazoxane (600 mg/m2 by IV infusion over 15 minutes), followed 15 minutes later by doxorubicin (60 mg/m2 IV), followed 15 minutes later by paclitaxel (150 or 175 mg/m2 by IV infusion over 3 hours) in cohorts of three to six patients using a standard phase I design without (group A) and with (group B) granulocyte colony-stimulating factor (G-CSF). Treatment continued until there was a substantial decrease in the left ventricular ejection fraction (LVEF), congestive heart failure, progressive disease, or physician discretion to discontinue. RESULTS: The MTD of paclitaxel was 150 mg/m2, and adjunctive therapy with G-CSF was required to prevent febrile neutropenia. Dexrazoxane had no significant effect on the pharmacokinetics of paclitaxel or doxorubicin. After a median cumulative doxorubicin dose of 360 mg/m2 (range, 60 to 870 mg/m2), no patient developed congestive heart failure or had a decrease in LVEF below normal. An objective response occurred in all five patients with locally advanced breast cancer and in eight of 20 patients (40%; 95% confidence interval, 19% to 61%) with metastatic breast cancer. CONCLUSION: When combined with doxorubicin (60 mg/m2) and dexrazoxane (600 mg/m2), paclitaxel given as a 3-hour infusion had an MTD of 150 mg/m2, and G-CSF was required to prevent febrile neutropenia. Dexrazoxane had no effect on the pharmacokinetics of paclitaxel or doxorubicin. No patient in this trial had a decrease in the LVEF below normal, compared with about 20% to 50% of patients treated with doxorubicin and paclitaxel without dexrazoxane in other trials.

Effect of prolonged topotecan infusion on topoisomerase 1 levels: a phase I and pharmacodynamic study.

Topoisomerase 1 (topo-1) inhibitors act on the target enzyme by forming "cleavable complex," a high molecular weight DNA protein adduct. The formation of such cleavable complexes results in depletion of the Mr 100,000 "free" topo-1 band detectable by Western blot. The objectives of this study were to determine the maximally tolerated dose of prolonged topotecan infusion in previously untreated and minimally pretreated patients. A secondary objective was to measure the effect of prolonged topotecan infusion on topo-1 levels in peripheral blood mononuclear cells (PBMCs) as a pharmacodynamic end point. In a prior Phase I study of 21-day topotecan infusion (H. Hochster et al., J. Clin. Oncol., 12: 553-559, 1994), the maximum tolerated dose for patients treated previously was 0.53 mg/m2/day for 21 days every 28 days. In this study, patients with no prior therapy were treated similarly at 0.7 mg/m2/day for 21 days, and doses were escalated in 0.1 mg/m2/day increments. Patients who had one prior chemotherapy regimen or radiation therapy to a portal of