SFC/FTIR, SFC/APCI-MS and MALDI-TOF-MS for the analysis of siloxane-ethylene oxide copolymers.

Polydimethylsiloxanes (PDMSs) modified by introducing ethylene oxide units with the aim of forming sufficiently water-soluble siloxane compounds were characterized using supercritical fluid chromatography (SFC) coupled with Fourier transform infrared (FTIR) spectroscopy and atmospheric pressure chemical ionization mass spectrometry (APCI-MS), and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS). SFC has a domain in analyzing oligomers. Hyphenated techniques enable elucidation of the components. Remarkable is the resolution and short analysis time of MALDI-TOF-MS. SFC also allows quantification of the basic and reaction products.

A two-part phase I trial of high-dose interleukin 2 in combination with soluble (Chinese hamster ovary) interleukin 1 receptor.

Our purpose was to determine the maximum tolerated dose and toxicity associated with soluble Chinese hamster ovary [s(CHO)] recombinant human interleukin (IL) 1 receptor (IL-1R; Immunex, Seattle, WA) administration in humans and to determine the effective biological dose and/or maximum tolerated dose of the s(CHO) IL-1R in combination with high-dose IL-2 as determined by reduction in IL-2 toxicity and modulation of its biological effects. Twenty-seven patients with metastatic cancer were treated with escalating doses of s(CHO) IL-1R at 1, 1, 5, 10, 20, 40, and 55 mg/m2 i.v. on days -6 (except cohort 2), 1, and 15 and IL-2 at doses of 300,000 IU/kg (cohort 1) and 600,000 IU/kg (cohorts 2-7) i.v. every 8 h on days 1-5 and 15-19. No toxicity directly attributable to s(CHO) IL-1R was observed. The median number of IL-2 doses was 23. Hypotension and neurotoxicity were the major dose-limiting toxicities for the IL-2/s(CHO) IL-1R combination. Of the 24 patients treated with full-dose IL-2, there were six responses, three complete and three partial (response rate, 25%). Three patients developed thyroid dysfunction, and all 3 responding melanoma patients exhibited vitiligo. The t1/2 of s(CHO) IL-1R alone was 24-30 h and was not significantly altered by coadministration with IL-2. Whole-blood functional assays indicated that sufficient s(CHO) IL-1R was present in the circulation at top dose levels to inhibit the in vitro effects of IL-1beta on IL-8 induction; however, no effect on IL-2-induced IL-8 induction, or on the IL-1beta- or IL-2-induced tumor necrosis factor production, was observed. Suppression of IL-2-mediated tumor necrosis factor alpha and IL-6 induction in vivo during the first 24 h after IL-2 administration was observed, and the neutrophil chemotactic defect normally seen with IL-2 was not observed. IL-1R antagonist induction far exceeded that seen previously with IL-2 alone. No inhibition of either serum C-reactive protein induction or enhanced urinary nitrate excretion and no consistent effect on IL-2-related changes in peripheral blood mononuclear cell phenotype or endothelial adhesion molecule expression were seen. The coadministration of s(CHO) IL-1R produced no apparent reduction in IL-2 clinical toxicity manifested by either the ability to administer more IL-2 than anticipated or a reduction in the toxicity associated with a given amount of IL-2. Therefore, no effective biological dose could be identified for the s(CHO) IL-1R.

Randomized placebo-controlled clinical trial of high-dose interleukin-2 in combination with a soluble p75 tumor necrosis factor receptor immunoglobulin G chimera in patients with advanced melanoma and renal cell carcinoma.

PURPOSE: A randomized, double-blind, placebo-controlled trial was performed to compare the toxicity and biologic effects of treatment with high-dose intravenous (IV) bolus interleukin-2 (IL-2) plus the recombinant human soluble p75 tumor necrosis factor (TNF) receptor immunoglobulin G (IgG) chimera (rhuTNFR:Fc) with high-dose IL-2 alone in patients with advanced melanoma and renal cell carcinoma. PATIENTS AND METHODS: Twenty patients with advanced melanoma or renal cell carcinoma were randomized to receive IL-2 (Chiron, Emeryville, CA) 600,000 IU/kg every 8 hours on days 1 to 5 and 15 to 19 (maximum, 28 doses) combined with placebo or the rhuTNFR:Fc fusion protein (Immunex, Seattle, WA) 10 mg/m2 on days 1 and 15 and 5 mg/m2 on days 3, 5, 17, and 19. The impact of rhuTNFR:Fc on IL-2 toxicity and biologic effects was evaluated. RESULTS: No clinically significant difference in toxicity was observed in the two treatment arms. The adjusted median number of IL-2 doses administered during cycle 1 was 24.5 (range, seven to 28) and 21.5 (range, five to 27) for the placebo and rhuTNFR:Fc arms, respectively (P = .544). IL-2-induced TNF bioactivity, neutrophil chemotactic defect, and serum IL-6, IL-8, and IL-1 receptor antagonist (IL-1RA) induction were suppressed by rhuTNFR:Fc. Two of nine assessable patients (22%) on IL-2/placebo and three of 10 patients (30%) on IL-2/rhuTNFR:Fc responded. CONCLUSION: Despite evidence of in vitro neutralization of TNF functional activity and partial inhibition of other secondary biologic effects of IL-2, rhuTNFR:Fc does not reduce the clinical toxicity associated with high-dose IL-2 therapy. These results suggest that the toxicity and antitumor effects of IL-2 treatment are independent of circulating TNF.

Phase I trial of interleukin 2 in combination with the soluble tumor necrosis factor receptor p75 IgG chimera.

Our purpose was to determine the effective biological dose and/or maximum tolerated dose of recombinant human tumor necrosis factor receptor:IgG chimera (rhuTNFR:Fc; Immunex, Seattle, WA) in combination with interleukin 2 (IL-2) with regard to reduction in IL-2 toxicity and modulation of biological effects of high-dose IL-2 administration. Twenty-four patients with metastatic cancer were treated with escalating doses of rhuTNFR:Fc at 1, 1, 5, 10, and 20 mg/m2 i.v. on days 1 and 15 (dose levels 1-5) or 10, 20, and 30 mg/m2 days 1 and 15 plus 50% dose on days 3, 5, 17, and 19 (dose levels 6-8) prior to IL-2 at doses of 300,000 IU/kg (dose level 1) and 600,000 IU/kg (dose levels 2-8) i.v. every 8 h on days 1-5 and 15-19. The t1/2 of rhuTNFR in patients receiving IL-2 was 72 h. The median number of IL-2 doses was 24, and central nervous system, skin, and cardiac arrhythmias were the major dose-limiting toxicities. TNF bioactivity was inhibited, and the polymorphonuclear leukocyte chemotactic defect normally seen with IL-2 was not observed. Increases in C-reactive protein, IL-6, IL-8, and IL-1 receptor antagonist levels were partially suppressed relative to historical controls, whereas peripheral blood mononuclear cell phenotypes, urinary nitrate, endothelial adhesion molecule expression in skin biopsies, and cellular infiltrates in tumor biopsies were consistent with findings in patients treated with IL-2 alone. Four patients developed thyroid dysfunction. There were five responses: two complete responses (both melanoma) and three partial responses (response rate, 21%). rhuTNFR:Fc may modulate the toxicity and some of the biological effects of IL-2 while preserving antitumor activity. Dose level 6 (10 mg/m2 on days 1 and 15, and 5 mg/m2 on days 3, 5, 17, and 19) has been chosen for a randomized, double-blind, placebo-controlled trial of IL-2 with and without rhuTNFR:Fc.

Interleukin-6-associated anemia: determination of the underlying mechanism.

Recombinant human interleukin-6 (rhIL-6) is a pluripotent cytokine with proinflammatory, antitumor, and growth factor effects. Clinical investigations of rhIL-6 either alone as immunotherapy or as a colony-stimulating factor in conjunction with chemotherapy have shown a dose-dependent, rapid onset, and largely reversible decrease in venous hematocrit levels. In an effort to determine the mechanism for the rhIL-6-associated anemia, we measured red blood cell volume serially in patients receiving rhIL-6 at either 30 micrograms/kg/day as a 120-hour continuous intravenous infusion (renal cell carcinoma) or 100 micrograms/kg/d intravenously over 1 hour for 5 days (melanoma) as part of two separate phase II trials. Radioisotope dilution assays with 51Cr-labeled autologous red blood cells and hemolysis screens were performed on day 1 before the initiation of therapy and on day 5 shortly before the end of therapy. In the 6 patients studied, the mean decrease in hemoglobin concentration was 1.9 +/- 0.94 g/dL. The mean decrease in the hematocrit level was 6% +/- 2% and the mean increase in total blood volume was 731 +/- 337 mL. These changes were explained by a mean decrease in red blood mass of 106 +/- 109 mL and a mean increase in plasma volume of 743 +/- 289 mL. The decrease in red blood cell mass was largely explained by phlebotomy during the hospitalization, but was not statistically significant (paired t-test, P = .06). All other changes were statistically significant (P < .05). Simple regression analysis indicated that the decrease in hematocrit level and increase in plasma volume were related (y = -1.78 - .0066X; R = -.74). Measurements of lactate dehydrogenase, bilirubin, haptoglobin, and reticulocyte counts and serial stool hemoccults did not indicate hemolysis or blood loss. We conclude that the anemia caused by IL-6 is caused by an increase in plasma volume.

Multiinstitutional phase II trial of intensive combination chemoimmunotherapy for metastatic melanoma.

PURPOSE: To evaluate the activity and toxicity of combined high-dose cisplatin, dacarbazine (DTIC), and tamoxifen chemotherapy and high-dose bolus interleukin-2 (IL-2) in patients with metastatic melanoma. PATIENTS AND METHODS: Patients with metastatic melanoma, Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and normal organ function were enrolled onto this multiinstitutional Cytokine Working Group trial. Patients received intensive chemoimmunotherapy consisting of cisplatin (50 mg/m2) and DTIC (350 mg/m2) intravenously (IV) on days 1 to 3 and 43 to 45, IL-2 600,000 IU/kg IV every 8 hours on days 12 to 16 and 26 to 30 (maximum, 28 doses), and tamoxifen 20 mg orally each day. Patients were evaluated for response at day 63 of each cycle, and responding patients were given a second cycle of therapy beginning on day 71 to 85. RESULTS: Thirty-eight patients were entered onto this study. Toxicities were as expected for the chemotherapy and immunotherapy components of this regimen. Overlapping toxicity consisted primarily of thrombocytopenia (76% of patients required platelet transfusions), neutropenia, anemia, fatigue, and weight loss. Despite these cytopenias, bleeding and infectious complications were rare. There were no treatment-related deaths. Three patients achieved a complete response (CR; 8%), and 13 achieved a partial response (PR). The overall objective response rate was 42% (95% confidence interval [CI], 26% to 58%). Six additional patients had greater than 50% tumor reduction at day 63, which did not persist until a subsequent evaluation. The median duration of response was 5 months (range, 2 to 20+), and the median survival duration was 11 months. CONCLUSION: This intensive treatment regimen appears to possess activity in metastatic melanoma comparable, but not superior, to that of other less intensive cisplatin- and IL-2-based chemoimmunotherapy regimens. Although the toxicity and complexity of this regimen make it unsuitable for phase III testing and impractical for more widespread use, the results of this study support a potential favorable interaction between IL-2 and chemotherapy in this disease and highlight the need for appropriately designed phase III trials.

Cyclic guanosine monophosphate production in the pituitary: stimulation by C-type natriuretic peptide and inhibition by gonadotropin-releasing hormone in alpha T3-1 cells.

Atrial, brain-type, and C-type natriuretic peptides (ANP, BNP, and CNP) act via receptors with intrinsic guanylate cyclase activity. The A-type and B-type ANP receptors are selectively activated by ANP and CNP, respectively. The anterior pituitary is a site of ANP production and action, suggesting a local regulatory function, and this may also hold true for CNP which is found at its highest tissue concentrations in the anterior pituitary. Here we show that these peptides all cause dose-dependent increases in cGMP accumulation in alpha T3-1 cells (a gonadotrope-derived cell line), GH3 cells, and in primary cultures of rat pituitary cells. The response to CNP is particularly robust in alpha T3-1 cells (59 +/- 9-fold increase, EC50 14 +/- 3 nM), and the rank order of potency in alpha T3-1 cells and primary cultures (CNP >> ANP > BNP) is suggestive of action exerted via B-type receptors. Although CNP did not alter GnRH-stimulated LH release or [3H]inositol phosphate accumulation, GnRH reduced CNP-stimulated cGMP accumulation dose dependently (EC50 approximately 0.1 nM). This inhibition reflects the ability of GnRH to shift the CNP dose-response curve rightward (increasing the EC50 for CNP action approximately 10-fold both with and without 3-isobutyl-1-methylxanthine). The inhibitory effect was not blocked by omission of extracellular Ca++ nor mimicked by the Ca++ ionophore A23187 but was mimicked by a protein kinase C (PKC)-activating phorbol ester (which had a comparable effect to GnRH on the EC50 for CNP action). The data imply that CNP rather than (or in addition to) ANP may have a local regulatory function within the pituitary, that although its role is currently unknown it may involve functional interaction with GnRH in gonadotropes, and that the effect of GnRH on CNP action may be PKC-mediated. Moreover, we suggest that alpha T3-1 cells may be a useful model for investigation of the cross-talk between the B-type natriuretic peptide receptor-regulated signal transduction pathway and the Ca++/PKC pathway activated by ligand-stimulated hydrolysis of inositol phospholipids.

Phase I evaluation of thrice-daily intravenous bolus interleukin-4 in patients with refractory malignancy.

PURPOSE: A phase I dose-escalation trial of recombinant human interleukin-4 (IL-4) was performed to determine its toxicity, biologic activity, and potential antineoplastic effects. PATIENTS AND METHODS: Ten patients with refractory malignancies received IL-4 by bolus intravenous injection every 8 hours on days 1 to 5 and 15 to 19 (maximum, 28 doses) of a 31-day study period. Three patients received 10 micrograms/kg per dose and seven received 15 micrograms/kg per dose of IL-4. RESULTS: Toxic symptoms noted at the second dose level included nasal congestion, diarrhea, nausea and vomiting, fatigue, anorexia, headache, dyspnea, and capillary leak syndrome (median weight gain, 6.1%; range, 3.4% to 11.7%). Fever or sustained hypotension sufficient to require pressors did not occur. Decreases in lymphocyte count and serum bicarbonate, sodium, albumin, fibrinogen and immunoglobulin (Ig) levels, and increases in hematocrit, prothrombin time/partial thromboplastin time (PT/PTT), soluble CD23, and, occasionally, serum creatinine and transaminases occurred. All side effects resolved by day 31. Phenotypic analysis of peripheral-blood mononuclear cells (PBMC) showed a decrease in the percentage of circulating CD16 and CD14(+) cells. Plasma tumor necrosis factor (TNF) and IL-1 beta levels were unaffected, whereas serum C-reactive protein (CRP) concentrations increased slightly and plasma IL-1 receptor antagonist (IL-1RA) levels increased markedly. No tumor responses were observed. CONCLUSIONS: We conclude that 10 micrograms/kg per dose of IL-4 is the maximum-tolerated dose for this schedule, although 15 micrograms/kg per dose can be tolerated if more intensive, but still non-intensive care unit level care is provided. The results of this study should aid in the design of future phase II trials that involve IL-4 alone or phase I studies that combine IL-4 with other cytokines such as IL-2.