Modulation of the random migration of human platelets.

Random migration of human platelets has been recognized as a parameter of platelet function which can be assessed in a reproducible manner by modification of the Boyden micropore filter technique for evaluating this function in other cells (Boyden, S. 1962. J. Exp. Med. 115: 453-466). Because platelets are extremely susceptible to aggregation, the conditions for collecting and isolating platelets and the migration buffer (Ca(++) and Mg(++)-free phosphate buffered saline, pH 6.8, with glucose and gelatin) were selected to minimize such a possibility. The random movement of platelets into the micropore filter was maximal at 30-37 degrees C and was contingent upon the metabolic integrity of the cell; thus, it can be attributed to active spontaneous migration. While the initiating and enhancing effects of epinephrine on the platelet aggregation-release reaction are mediated by an alpha-adrenergic receptor, the inhibition of random migration involved a beta-receptor. Equimolar propranolol but not phentolamine prevented epinephrine inhibition of random migration, and isoproterenol had activity comparable to epinephrine while phenylephrine was inactive. The capacity of the cholinomimetic agent, carbachol, to increase platelet migration is reminiscent of the recent findings in several cell systems in which beta-adrenergic and cholinergic stimuli have opposite effects. The prostaglandins E1 and E2 augmented spontaneous migration in contrast to their well established inhibitory action on platelet aggregation at the concentrations employed. The suppression by indomethacin of prostaglandin enhancement and of spontaneous migration implies a requirement for the prostaglandin biosynthetic pathway during the migration process. Thus, the spontaneous migration of human platelets, an additional parameter of platelet function for in vitro investigations, disclosed not only a beta-adrenergic receptor for epinephrine, but also a capacity for cholinergic augmentation and an apparent requirement for prostaglandin biosynthesis.

Specific inhibition of the polymorphonuclear leukocyte chemotactic response to hydroxy-fatty acid metabolites of arachidonic acid by methyl ester derivatives.

The human polymorphonuclear (PMN) leukocyte chemotactic activity of the hydroxy-fatty acid metabolites of arachidonic acid, 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-l-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), is eliminated by methylation. Both methyl esters are specific competitive inhibitors of the PMN leukotactic responses to the parent stimuli, and exert no effect on the responses to formyl-methionyl peptides or chemotactic fragments of the fifth component of complement. 50% inhibition of the in vitro chemotactic responses of PMN leukocytes to HETE and HHT was achieved by an equimolar concentration of the corresponding methyl esters, whereas reciprocal cross-inhibition was observed at molar ratios of HETE methyl ester to HHT and HHT methyl ester to HETE which reflected the three- to fivefold greater chemotactic potency of HETE relative to HHT. Methyl esters of structurally related, but nonchemotactic, fatty acids did not competitively inhibit the chemotaxis elicited by HETE or HHT. The intraperitoneal injection of HETE in guinea pigs evoked an eosinophil response at 30 min and a neutrophil response at 5 h, which were prevented by a one-to twofold molar ratio of HETE methyl ester. The competitive inhibition of the in vitro chemotactic activity and the in vivo leukotactic effect of the unsaturated hydroxy-fatty acids by homologous methyl ester derivatives suggests that the cellular component of natural inflammatory reactions may be susceptible to specific regulation by receptor-directed modulation of the activity of the predominant chemotactic principles.

Quantitation of arachidonic acid lipoxygenase products in malignant and nonmalignant effusions.

The arachidonic acid lipoxygenase products 15-hydroxyeicosatetraenoic acid, 12-hydroxyeicosatetraenoic acid, and 5-hydroxyeicosatetraenoic acid and the arachidonic acid cyclooxygenase product prostaglandin E2 were quantitated in 11 malignant and 10 nonmalignant peritoneal and pleural effusions. The mean quantities of 15-hydroxyeicosatetraenoic acid in malignant and nonmalignant effusions were 10 and 46 ng/ml, respectively (p less than 0.05), and the mean quantities of 5-hydroxyeicosatetraenoic acid in malignant and nonmalignant effusions were less than 1 ng/ml and 12 ng/ml, respectively (p less than 0.05). The quantities of 12-hydroxyeicosatetraenoic acid and prostaglandin E2 were also reduced in malignant effusions but the reduction was not statistically significant. Both the protein content and the total number of leukocytes correlated with the quantity of prostaglandin E2 in the effusions, whereas no consistent correlation was found with the quantity of lipoxygenase products in the effusions. Lipoxygenase products are critical mediators of neutrophil, lymphocyte, and macrophage function. Thus, the reduced capacity to lipoxygenate arachidonic acid associated with malignant conditions may contribute to the impaired immune responsiveness of patients with cancers.

Potentiation of mitogen-induced human T-lymphocyte activation by retinoic acid.

The capacity of retinoic acid to modulate human T-lymphocyte and B-lymphocyte activation by mitogens was examined. T-lymphocyte proliferation stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) or phytohemagglutinin was enhanced by 5 nM to 5 microM retinoic acid in a dose-dependent manner with a 65 +/- 35% (SD) increase (n = 6, P less than 0.01) in TPA-stimulated proliferation induced by 5 microM retinoic acid. Retinoic acid enhanced T-lymphocyte proliferation over a wide range of background proliferation induced by different TPA concentrations. Retinoic acid alone did not stimulate T-lymphocyte proliferation. In contrast retinoic acid inhibited B-lymphocyte proliferation stimulated by TPA or phytohemagglutinin with 26.7 +/- 23.4% inhibition of TPA-stimulated proliferation induced by 5 microM retinoic acid (P less than 0.02). Retinoic acid had intermediate effects on the proliferation of different mixtures of T- and B-lymphocytes stimulated by TPA or phytohemagglutinin. The recognition that retinoic acid has opposing effects on human T- and B-lymphocyte activation by mitogens may account for the conflicting reports of the effects of retinoids on the immune response of unpurified human lymphocyte preparations.

Enhanced arachidonic acid lipoxygenation by K562 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate.

The arachidonic acid lipoxygenase products released into culture supernatants by the human myeloid cell line K562 were quantitated by high-performance liquid chromatography. During 2 hr of incubation, K562 cells spontaneously released a mean of 9 ng of 15-monohydroxyeicosatetraenoic acid, 35 ng of 5-monohydroxyeicosatetraenoic acid, and 87 ng of a partially resolved mixture of 11- and 12-monohydroxyeicosatetraenoic acid per 10(7) cells. The addition of 50 micrograms of arachidonic acid per ml to the cell cultures increased the quantities of monohydroxyeicosatetraenoic acids generated after 2 hr of incubation by 10- to 100-fold; changes in the ratios of these lipoxygenase products occurred over 24 hr of incubation. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate, at concentrations of 5 to 25 ng/ml increased arachidonic acid lipoxygenation 1- to 2-fold in cultures containing 50 micrograms of arachidonic acid per ml, and up to 20-fold in cultures not supplemented with arachidonic acid. The lipoxygenation of arachidonic acid was enhanced 2- to 9-fold for up to 24 hr after a 2-hr exposure of K562 cells to 12-O-tetradecanoylphorbol-13-acetate. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked the effects of 12-O-tetradecanoylphorbol-13-acetate, suggesting that the monohydroxyeicosatetraenoic acids are the products of specific lipoxygenases rather than of nonenzymatic oxidative reactions. The capacities of phorbol esters to promote tumors in the mouse skin model corresponded to their respective capacities to enhance the lipoxygenation of arachidonic acid to 15- and 5-monohydroxyeico-satetraenoic acid but not to 11- and 12-monohydroxyeico-satetraenoic acid.

Immunotherapy of hormone-refractory prostate cancer with antigen-loaded dendritic cells.

PURPOSE: Provenge (Dendreon Corp, Seattle, WA) is an immunotherapy product consisting of autologous dendritic cells loaded ex vivo with a recombinant fusion protein consisting of prostatic acid phosphatase (PAP) linked to granulocyte-macrophage colony-stimulating factor. Sequential phase I and phase II trials were performed to determine the safety and efficacy of Provenge and to assess its capacity to break immune tolerance to the normal tissue antigen PAP. PATIENTS AND METHODS: All patients had hormone-refractory prostate cancer. Dendritic-cell precursors were harvested by leukapheresis in weeks 0, 4, 8, and 24, loaded ex vivo with antigen for 2 days, and then infused intravenously over 30 minutes. Phase I patients received increasing doses of Provenge, and phase II patients received all the Provenge that could be prepared from a leukapheresis product. RESULTS: Patients tolerated treatment well. Fever, the most common adverse event, occurred after 15 infusions (14.7%). All patients developed immune responses to the recombinant fusion protein used to prepare Provenge, and 38% developed immune responses to PAP. Three patients had a more than 50% decline in prostate-specific antigen (PSA) level, and another three patients had 25% to 49% decreases in PSA. The time to disease progression correlated with development of an immune response to PAP and with the dose of dendritic cells received. CONCLUSION: Provenge is a novel immunotherapy agent that is safe and breaks tolerance to the tissue antigen PAP. Preliminary evidence for clinical efficacy warrants further exploration.

Phase Ia/Ib trial of bispecific antibody MDX-210 in patients with advanced breast or ovarian cancer that overexpresses the proto-oncogene HER-2/neu.

PURPOSE: MDX-210 is a bispecific antibody that binds simultaneously to type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI) and to the HER-2/neu oncogene protein product. MDX-210 effectively directs Fc gamma RI-positive effector cells such as monocytes and macrophages to phagocytose or kill tumor cells that overexpress HER-2/neu. The goals of this phase Ia/Ib trial were to determine the maximum-tolerated dose (MTD) and/or the optimal biologic dose (OBD) of MDX-210. PATIENTS AND METHODS: Patients with advanced breast or ovarian cancer that overexpressed HER-2/neu were eligible for treatment. Cohorts of three patients received a single intravenous (IV) infusion of MDX-210 at increasing dose levels from 0.35 to 10.0 mg/m2. RESULTS: Treatment was well tolerated, with most patients experiencing transient grade 1 to 2 fevers, malaise, and hypotension only. Two patients experienced transient grade 3 hypotension at 10.0 mg/m2. Transient monocytopenia and lymphopenia developed at 1 to 2 hours, but no other hematologic changes were observed. Doses of MDX-210 > or = 3.5 mg/m2 saturated > or = 80% of monocyte Fc gamma RI and produced peak plasma concentrations > or = 1 microgram/mL, which is greater than the concentration for optimal monocyte/macrophage activation in vitro. Elevated plasma levels of the monocyte products tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), and neopterin were observed with maximal levels at doses > or = 7.0 mg/m2. Localization of MDX-210 in tumor tissue was demonstrated in two patients. One partial and one mixed tumor response were observed among 10 assessable patients. CONCLUSION: MDX-210 is immunologically active at well-tolerated doses. The MTD and OBD is 7 to 10 mg/m2.

Treatment of patients with advanced colorectal carcinomas with fluorouracil alone, high-dose leucovorin plus fluorouracil, or sequential methotrexate, fluorouracil, and leucovorin: a randomized trial of the Northern California Oncology Group.

We compared the effectiveness of fluorouracil (5-FU) alone (arm A), high-dose leucovorin plus 5-FU (arm B), and sequential methotrexate, 5-FU, and leucovorin (arm C) for treatment of patients with advanced colorectal carcinomas who had not received prior chemotherapy. Arm A consisted of infusions of 5-FU at 12 mg/kg/d intravenously (IV) for 5 days followed by weekly infusions of 5-FU at 15 mg/kg; arm B consisted of leucovorin infusions at 200 mg/m2/d IV plus infusions of 5-FU at 400 mg/m2/d IV on days 1 through 5 of a 28-day cycle; arm C consisted of methotrexate at 50 mg/m2 orally every 6 hours for five doses followed by infusions of 5-FU, 500 mg/m2 IV, and leucovorin, 10 mg/m2 orally, every 6 hours for five doses every other week. A total of 265 patients were entered into the trial, of whom 249 (94%) were fully evaluable. The objective response rate (complete [CR] plus partial [PR] responses) was 17.3% on arm A, 18.8% on arm B, and 19.8% on arm C (log-rank test, P greater than .4). The median time to failure was 138 days on arm A, 166 days on arm B, and 182 days on arm C (log-rank test, P values of arm A v B = .06; arm A v arm C = .04). Median survival was 345 days on arm A, 324 days on arm B, and 356 days on arm C (log-rank test, P greater than .4). Treatment with 5-FU alone was significantly more dose intensive and more toxic than either of the experimental combinations. The rates of grade 3 or greater nonhematologic toxicity were 42.3% on arm A, 24.3% on arm B, and 14.3% on arm C. Hematologic toxicity was milder but had the same pattern. This study indicates that these regimens of high-dose leucovorin plus 5-FU and sequential methotrexate, 5-FU, and leucovorin are not more effective than is 5-FU alone for treatment of patients with colorectal carcinomas when 5-FU is administered at high-dose intensity.

Fluorouracil modulation in colorectal cancer: lack of improvement with N -phosphonoacetyl- l -aspartic acid or oral leucovorin or interferon, but enhanced therapeutic index with weekly 24-hour infusion schedule--an Eastern Cooperative Oncology Group/Cancer and Leukemia Group B Study.

PURPOSE: To investigate mechanism-directed regimens in maximizing the efficacy of fluorouracil (5-FU) in advanced colorected cancer. PATIENTS AND METHODS: Based on promising phase II data, a randomized comparison of various methods for the biochemical modulation of 5-FU was undertaken in patients with advanced colorectal cancer. The control group received single-agent 5-FU as a 24-hour infusion weekly. Patients (N = 1,120) with no prior chemotherapy for metastatic disease were randomized to one of the following arms: arm A, 5-FU 2,600 mg/m2 by 24-hour infusion, weekly; arm B, N-phosphonoacetyl-l-aspartic acid 250 mg/m2 day l, 5-FU 2,600 mg/m2 by 24-hour infusion day 2, weekly; arm C, 5-FU 600 mg/m2 with oral leucovorin (LV) 125 mg/m2 hourly for the preceding 4 hours, weekly; arm D, 5-FU 600 mg/m2 with intravenous (IV) LV 600 mg/m2, weekly; arm E, 5-FU 750 mg/m2/d IV by continuous infusion for 5 days, then 750 mg/m2 weekly, and recombinant interferon alfa-2a 9 million units subcutaneously three times weekly. Median follow-up was 4.8 years. RESULTS: Of the 1,098 assessable patients, 57% had measurable disease. The toxicity of all the regimens was tolerable. Grade 4 or worse toxicity occurred in 11%, 11%, 30%, 24%, and 22% on each arm, respectively; diarrhea was the most common adverse effect. These toxicity patterns favored significantly (P <.001) the 24-hour infusion arms. Median survival (months) by arm was A, 14.8; B, 11.9; C, 13.5; D, 13.6; and E, 15.2. These survival durations did not differ significantly. CONCLUSION: We conclude that a weekly infusion regimen of 5-FU is significantly less toxic than and as effective as 5-FU bolus regimens modulated by either LV or interferon in patients with metastatic colorectal cancer.

Priming tissue-specific cellular immunity in a phase I trial of autologous dendritic cells for prostate cancer.

We attempted to induce therapeutic immunity against prostate-derived tissues in patients suffering from progressive hormone-refractory metastatic prostate carcinoma. Thirteen patients were treated with two infusions, 1 month apart, of autologous dendritic cells (APC8015) preexposed ex vivo to PA2024, a fusion protein consisting of human granulocyte/macrophage-colony stimulating factor (GM-CSF) and human prostatic acid phosphatase (PAP). The infusions were followed by three s.c. monthly doses of PA2024 without cells. Three groups of patients each received PA2024 at 0.3, 0.6, or 1.0 mg/injection. All Ps were two-sided. Treatment was well tolerated. After infusions of APC8015, patients experienced only mild (grade 1-2) short-lived fever and/or chills, myalgia, pain, and fatigue. One patient developed grade 3 fatigue. Four patients developed mild local reactions to s.c. PA2024. Twelve patients were evaluable for response to treatment. Circulating prostate-specific antigen levels dropped in three patients. T cells, drawn from patients after infusions of APC8015, but not before, could be stimulated in vitro by GM-CSF (P = 0.0004) and PAP (P = 0.0001), demonstrating broken immune tolerance against these two normal proteins. Injections of PA2024 did not influence the reactivity of T cells against PAP and GM-CSF. However, antibodies to GM-CSF and, to a much lesser extent, to PAP reached maximum titers only after two or even three injections of PA2024, showing that directly injected PA2024 was involved in stimulation of humoral immunity. Dendritic cells exposed to antigen ex vivo can induce antigen-specific cellular immunity in prostate cancer patients, warranting further studies of this mode of immunotherapy.

Interleukin-2, cisplatin, and 5-fluorouracil for patients with non-small cell lung and head/neck carcinomas.

The feasibility and efficacy of treating patients with locally recurrent or metastatic non-small cell lung cancer (NSCLC) or head/neck cancer with interleukin-2 (IL-2), cisplatin, and 5-fluorouracil (5-FU) was tested. Treatment was given every 28 days and consisted of cisplatin, 100 mg/m2 on days 1 and 8; 5-FU, 1,000 mg/m2 by continuous infusion on days 1-3; and IL-2, 12 million units/m2 by i.v. bolus on days 15-19. Thirty-four patients (22 NSCLC, 12 head/neck cancer) were registered in the study. The median age was 58 years; 59% had Karnofsky performance status of 70-80% and over one-half received prior therapy. All patients were evaluable for toxicity and 29 (18 NSCLC, 11 head/neck cancer) were evaluable for response. Twenty-five patients experienced at least one grade 3 or 4 toxicity, but these toxicities were transient and, in general, well tolerated. The response rate was 37% for NSCLC (0 complete response, 7 partial response) and 55% for head/neck cancer (2 complete response, 4 partial response). Two patients with head/neck cancer responded to treatment after failing prior therapy with cisplatin/5-FU alone. The combination of IL-2, cisplatin, and 5-FU is tolerable and active for treatment of NSCLC and head/neck carcinoma; the combination may not be cross-resistant with other chemotherapy combinations. Further studies of IL-2 combined with cisplatin/5-FU are warranted to determine the most effective dose and schedule.

Soluble normal and mutated DNA sequences from single-copy genes in human blood.

Healthy individuals have soluble (extracellular) DNA in their blood, and increased amounts are present in cancer patients. Here we report the detection of specific sequences of the cystic fibrosis and K-ras genes in plasma DNA from normal donors by amplification with the polymerase chain reaction. In addition, mutated K-ras sequences are identified by polymerase chain reaction utilizing allele-specific primers in the plasma or serum from three patients with pancreatic carcinoma that contain mutated K-ras genes. The mutations are confirmed by direct sequencing. These results indicate that sequences of single-copy genes can be identified in normal plasma and that the sequences of mutated oncogenes can be detected and identified with allele-specific amplification by polymerase chain reaction in plasma or serum from patients with malignant tumors containing identical mutated genes. Mutated oncogenes in plasma and serum may represent tumor markers that could be useful for diagnosis, determining response to treatment, and predicting prognosis.

Rapidly alternating radiotherapy and high dose cisplatin chemotherapy in stage IIIB non-small cell lung cancer: results of a phase I/II study.

Alternating radiotherapy and chemotherapy increases tumor cure rates in some animal models with reduced normal tissue damage compared to sequential use of these modalities. To test this concept in non-small cell lung cancer, 23 patients with predominantly Stage IIIB disease were treated on a Northern California Oncology Group pilot study of alternating radiotherapy and high dose cisplatin. Radiotherapy consisted of 6000 cGy delivered in three separate 10-day courses of 200 cGy/fraction/day during weeks 1 and 2, 5 and 6, and 9 and 10. High dose cisplatin, 100 mg/m2 in 3% saline, was administered on weeks 3 and 4, 7 and 8, 11 and 12, and 15 and 16. The response rate in 22 eligible patients is 73% (16/22) with four complete responses and 12 partial responses. Feasibility of this approach is demonstrated by 20/22 patients completing radiotherapy and a median of 2.5 courses of chemotherapy administered. Median survival time is 14.2 months (range 2-40+ months). One- and 2-year survival rates are 64% (14/22) and 41% (9/22), respectively. Hematologic, renal, and radiation-related toxicities were significant but manageable. We conclude that rapid alternation of radiotherapy and a high dose intensity cisplatin regimen is feasible in Stage IIIB non-small cell lung cancer, with a high response rate and acceptable toxicity. The long-term impact on local control and survival remains unclear, although preliminary survival data are encouraging in this poor prognosis population. Further studies of this concept are warranted.

A randomized phase II study of ifosfamide/mesna/cisplatin plus G-CSF or etoposide/cisplatin plus G-CSF in advanced non-small cell lung cancer: a Cancer and Leukemia Group B study.

This Phase II study was designed to determine the efficacy of two chemotherapy regimens with G-CSF support for patients with advanced non-small cell lung cancer (NSCLC). One-hundred and one patients with Stage IIIB or IV NSCLC and performance status 0-1 were randomized to receive ifosfamide 2.0 g/m2 days 1-3, mesna 400 mg/m2 at 0, 4, 6 h days 1-3, cisplatin 33 mg/m2 days 1-3 or etoposide 200 mg/m2 days 1-3, cisplatin 35 mg/m2 days 1-3. Both groups received G-CSF 5 micrograms/kg SQ day 4 to the post day 11 absolute neutrophil count > 10 000. For the 47 eligible patients receiving ifosfamide/mesna/cisplatin, the response rate was 26% (95% confidence interval: 14-40%) and the median survival 7.5 months (95% confidence interval: 5.8-11.0 months). Grade 3 or worse toxicities were: neutropenia 75%, thrombocytopenia 70%, infection 21%. There were two treatment-related deaths due to infection. For course 1, the median absolute neutrophil count nadir was 1.3, platelet nadir 96 000 and incidence of febrile neutropenia 16%. For the 48 eligible patients receiving etoposide/cisplatin, the response rate was 21% (95% confidence interval: 11-35%) and median survival 5.8 months (95% confidence interval: 4.5-9.7 months). Grade 3 or worse toxicities were: neutropenia 90%, thrombocytopenia 58%, infection 29%. There were three treatment-related deaths due to infection. For course 1, the median absolute neutrophil count was 0.2, platelet nadir 80 000 and incidence of febrile neutropenia 33%. For both ifosfamide/mesna/cisplatin and etoposide/cisplatin, median duration of Grade IV neutropenia was short (< or = 4 days), time to subsequent courses 21 days and dose delivered > 95% of planned dose. Although G-CSF allowed full doses of drugs to be delivered on schedule, both ifosfamide/mesna/cisplatin and etoposide/cisplatin produced response rates and survival similar to other cisplatin-based regimens. In view of the significant cost of G-CSF and no obvious improvement in response rate, survival or toxicity profile, G-CSF cannot be recommended with these chemotherapy regimens for patients with advanced NSCLC.

Synthesis of platelet activating factor by ocular tissue from inflamed eyes.

Platelet activating factors (PAFs) are a family of ether lipids with properties that suggest a major role in inflammation. We have previously implicated PAFs in ocular inflammation based on the inhibition of several rabbit models of iritis with a specific PAF receptor antagonist. We have tested ocular tissues for the ability to synthesize PAF. Iris, ciliary body, cornea, and/or retina were carefully dissected from New Zealand white rabbits, and tissue from four eyes was pooled. Tissues were stimulated with calcium ionophore (10 mumol/L), and supernatants were extracted with chloroform-methanol. Platelet-aggregating activity was found in the chloroform phase in 2 of 9, 1 of 8, 0 of 9, and 3 of 9 studies involving iris, retina, ciliary body, or cornea, respectively. Twenty-four hours after the intravitreal injection of 125 ng of endotoxin, aggregating activity was consistently detectable from supernatants of stimulated iris and ciliary body, occasionally present from stimulated retina but not detectable from cornea. The shape of the aggregation curve resembled that produced by 0.5 to 2.0 ng of authentic PAF. Moreover, the aggregation could be completely inhibited by a PAF receptor antagonist and the aggregating activity chromatographed identically on high-performance liquid chromatography to a PAF standard. These studies indicate that PAF-like activity could be detected from several ocular tissues subsequent to inflammation. Iris, ciliary body, retina, vascular endothelium, and/or leukocytes could each contribute to the presence of this inflammatory mediator.

Phase I trial of a 5-day infusion of L-leucovorin plus daily bolus 5-fluorouracil in patients with advanced gastrointestinal malignancies.

The combination of leucovorin [(6d,l)-5-formyl-tetrahydrofolate] and 5-fluorouracil (5-FU) has increased efficacy compared to 5-FU alone as treatment of advanced colorectal cancer. Leucovorin is metabolized to methylene tetrahydrofolate, which potentiates the antitumor actions of 5-FU by forming a ternary complex of thymidylate synthase, fluorodeoxyuridine and methylene tetrahydrofolate. Only l-leucovorin is metabolized to methylene tetrahydrofolate and forms this ternary complex. However, d-leucovorin may not be inert. d-Leucovorin may impair cellular uptake and metabolism of l-leucovorin, thereby inhibiting the actions of l-leucovorin. Because of this possible limitation to the effectiveness of racemic leucovorin, we have begun to explore the effects of the pure, biologically active isomer, l-leucovorin. In this phase I trial, patients with advanced gastrointestinal malignancies were treated with a 5-day continuous infusion of l-leucovorin and daily intravenous boluses of 5-FU at 370 mg/m2. The dose of l-leucovorin was escalated in groups of three patients at four doses, 200 mg/m2 per day, 400 mg/m2 per day, 700 mg/m2 per day and 1000 mg/m2 per day. Treatment was repeated every 28 days. Seventeen patients with advanced gastrointestinal cancers entered the trial. Sixteen patients were evaluable for toxicity. Toxicity was similar to that expected for leucovorin plus 5-FU. The most common severe toxicities (and the number of patents affected) were: diarrhea (2), mucositis (2), nausea/vomiting (1), and abdominal/rectal pain (2). The maximum tolerated dose of l-leucovorin was 700 mg/m2 per day. Twelve patients were evaluable for response. One complete, one partial and one minor response were observed. All responses occurred among the nine patients with colorectal carcinomas. The combination of l-leucovorin and 5-FU is well tolerated by patients and appears active for treatment of advanced colorectal carcinomas. Additional clinical trials are necessary to determine if l-leucovorin is more effective than d,l-leucovorin for modulating the effectiveness of 5-FU.

Inhibition of platelet-activating factor binding to human platelets by calcium channel blockers.

Calcium channel blockers may impair cell activation either by inhibiting calcium influx or by inhibiting agonist binding. Because of this dual action of calcium channel blockers and because of the close relationship between calcium influx and platelet-activating factor (PAF) binding to platelets the current studies examined the effect of calcium channel blockers on PAF binding to washed human platelets. Diltiazem and verapamil inhibited aggregation by PAF in a dose-dependent manner with 50% inhibition at 2.8 +/- 1.4 X 10(-5) M diltiazem (mean +/- SD, n = 5) and 4.2 +/- 2.0 X 10(-5) M verapamil. Both channel blockers also inhibited PAF binding in a dose-dependent manner with 50% inhibition at 4.7 +/- 2.5 X 10(-5) M diltiazem and 6.3 +/- 1.2 X 10(-5) M verapamil. Analysis of the mechanisms of inhibition of binding indicate both competitive and non-competitive effects of the channel blockers. Scatchard analysis of PAF binding in the presence of different fixed concentrations of either diltiazem or verapamil revealed that these agents both increased PAF receptor number and decreased the receptor binding affinity. Lineweaver-Burke analysis of the same data revealed a family of lines which intersect to the right of the ordinate. The channel blockers also dissociated previously-bound PAF from platelets. The current studies indicate that calcium channel blockers inhibit platelet activation by PAF by more than one mechanism and suggest that the PAF receptor may be closely associated with calcium channels.

Platelet-activating factor binding and metabolism during human platelet aggregation.

Platelet-activating factor (PAF) binding and metabolism were quantified during human platelet aggregation induced by [3H]PAF. Platelet aggregation was maximal within 45 to 75 sec. PAF binding was maximal within 15 to 60 sec and then remained stable for at least 20 min. Total binding during aggregation elicited by 1.2 pmol [3H]PAF was 13.4% +/- 1.0% (0.16 +/- 0.01 pmol/8 x 10(7) platelets) (mean +/- SEM) and specific, receptor binding was 4.3% +/- 0.3% (0.05 +/- 0.003 pmol/8 x 10(7) platelets). Specific binding was fully reversible at the time of maximal aggregation. Scatchard analysis of PAF binding at the time of maximal platelet aggregation revealed 124 +/- 56 receptors per platelet with a dissociation constant of 0.56 +/- 0.04 nM. Binding of 52 +/- 23 PAF molecules per platelet elicited maximal platelet aggregation (mean +/- SD). Less than 2% of the bound PAF was metabolized at the time of maximal aggregation and the principal metabolite was the inactive product lysoPAF. Increased metabolism to alkyl-acyl-glycero-3-phosphocholine was observed at later times. These studies indicate that PAF receptor internalization and PAF metabolism are not essential to human platelet activation by PAF.

Decay of the activating signal after platelet stimulation with 1-0-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine: changes in calcium permeability.

The decay of the platelet-activating signal after platelet stimulation with the platelet-activating factor 1-0-alkyl-2-acetyl-SN-glycero-3-phosphorylcholine (AGEPC) was examined. Washed human platelets in calcium-poor buffer were stimulated with AGEPC followed 0.25 to 10 min later by reconstitution with 1.8 mM calcium. AGEPC did not induce platelet aggregation in calcium-poor buffer but aggregation occurred upon addition of calcium. The capacity of calcium-reconstitution to elicit platelet aggregation decayed rapidly after platelet exposure to AGEPC (T 1/2 = 1 min). A parallel decay of AGEPC-induced increased calcium permeability (R = 0.93) was demonstrated using the fluorescent probe Quin 2. Studies with calcium channel blockers suggest that AGEPC opens a calcium channel which is distinct from those present in unstimulated platelets.

Bispecific Antibody MDX-210 for Treatment of Advanced Ovarian and Breast Cancer.

A large number of monoclonal antibodies (MAbs) to various tumor cell lines have been developed (1). However, MAbs have thus far had limited therapeutic impact in oncology, probably in part because many murine MAbs do not effectively recruit immune effector mechanisms, such as complement fixation and antibody-dependent cell-mediated cytotoxicity (ADCC) in humans. Additionally, although humanized MAbs are being developed, when used therapeutically their immunological effectiveness may be limited by high concentrations of nonspecific immunoglobulin (Ig) in patient serum. These nonspecific Ig will compete with conventional MAbs for binding to Type I Fc receptors (FcγRI) on immune effector cells, and may therefore limit conventional MAbs ability to recruit an immune response. Recently, however, clinical efficacy of a humanized MAb directed against HER-2/neu in patients with advanced breast cancer has been demonstrated (2-4). Preclinical data suggests that mechanistically this activity may be as a consequence of modulation of important biologic properties of the HER-2/neu receptor itself, as opposed to through an immunologic mechanism of tumor cell destruction.