ATP-dependent accumulation of CDCF in MRP2-expressing membrane vesicles: inhibition by oxaliplatin.

<p>MRP2-expressing and control membrane vesicles were incubated with CDCF (5 μM) for 5 min with or without ATP (4 mM) and oxaliplatin (400 μM), before measurement of CDCF accumulation by fluorescence. The <i>P</i> values shown as numbers are from two-way ANOVA and those shown as *** (<i>P</i>< 0.001) and N.S. (<i>P</i>> 0.05) are from Tukey’s multiple comparison post-tests following two-way ANOVA for comparisons with the respective membrane vesicles incubated with ATP but no oxaliplatin. Bars represent the means and standard deviations of individual values (open symbols) pooled from two independent experiments. Grey bars, MRP2-expressing membrane vesicles. Black bars, control membrane vesicles.</p

HPLC-UV detection of oxaliplatin, glutathione and degradation products in membrane vesicle incubation buffer.

<p>Injection of blank membrane vesicle incubation buffer (A) and authentic standards showed chromatographic separation of glutathione (B, peak 1, retention time 6.5 min), oxaliplatin (C, peak 2, retention time 12.5 min) and Pt(DACH)Cl₂ (D, peak 3, retention time 10.5 min) with no interference from components of the blank membrane vesicle incubation buffer. Oxaliplatin was incubated (100 μM, pH 7.4, 37˚C) in membrane vesicle incubation buffer with (I-L) or without glutathione (2 mM) (E-H) before analysis of samples by HPLC-UV after 0 hours (E,I), 0.3 hours (F,J), 2 hours (G,K) or 7 hours (H,L) incubation time. After 0.3 hours incubation time (F,J), HPLC-UV chromatograms appeared more-or-less unchanged from the start of the incubation (E,I) and similar with (J) and without glutathione (F). With an increasing incubation time, the oxaliplatin peak areas progressively reduced, and new peaks appeared, corresponding to Pt(DACH)Cl₂ (G,H: peak 3) in solutions containing no glutathione, and an unknown peak (K,L: peak 4) in solutions containing glutathione. Chromatograms are representative of those from two independent experiments.</p

MRP2-mediated membrane vesicle accumulation of oxaliplatin-derived platinum: dependence upon oxaliplatin exposure concentration and ATP.

<p>MRP2-expressing membrane vesicles were incubated with oxaliplatin (6.25 to 400 μM) with or without ATP (4 mM) for 10 min before measurement of platinum accumulation by ICPMS. The <i>P</i> values shown as numbers are from two-way ANOVA and those shown as **** (<i>P</i>< 0.0001) are from Tukey’s multiple comparison post-tests following two-way ANOVA. The bars represent means and standard deviations from individual values (open symbols) pooled from two independent experiments. Light grey bars, with ATP. Dark grey bars, without ATP.</p

Membrane vesicle accumulation of oxaliplatin-derived platinum: dependence upon MRP2, ATP and oxaliplatin exposure time.

<p>MRP2-expressing and control membrane vesicles were incubated with oxaliplatin (100 μM), with or without ATP (4 mM) for 5, 10 and 20 min, followed by measurement of platinum accumulation by ICPMS. The <i>P</i> values shown as numbers are from two-way ANOVA and those shown as *** (<i>P</i>< 0.001) and **** (<i>P</i> < 0.0001) are from Tukey’s multiple comparisons post-tests following two-way ANOVA for comparisons with MRP2-expressing membrane vesicles incubated with ATP at each respective time point. Bars represent means and standard deviations of individual values (open symbols) pooled from two independent experiments. Light grey bars, MRP2-expressing membrane vesicles with ATP. Dark grey bars, MRP2-expressing vesicles without ATP. Black bars, control membrane vesicles.</p

Effects of MRP2 inhibitors on membrane vesicle accumulation of oxaliplatin-derived platinum.

<p>MRP2-expressing and control membrane vesicles were incubated with oxaliplatin (100 μM), ATP (4 mM) and MRP2 inhibitors for 10 minutes before measurement of platinum accumulation by ICPMS. The <i>P</i> values shown as numbers are from two-way ANOVA and those shown as * (<i>P</i> < 0.05), ** (<i>P</i> < 0.01) and *** (<i>P</i> < 0.001) are from Tukey’s multiple comparison post-tests following two-way ANOVA for comparisons to MRP2-expressing membrane vesicles incubated with oxaliplatin and ATP but without MRP2 inhibitors. Bars represent means and standard deviations of individual values (open symbols) pooled from at least two independent experiments. Light grey bars, MRP2-expressing membrane vesicles. Black bars, control membrane vesicles.</p

Kinetic analysis of oxaliplatin degradation in membrane vesicle incubation buffer containing glutathione.

<p>Open symbols represent individual values of oxaliplatin chromatographic peak areas pooled from two independent experiments. The line represents a nonlinear one phase exponential decay regression fit (r² = 0.964) to the data giving an oxaliplatin degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours).</p

A Phase 1 Study of AS1409, a Novel Antibody-Cytokine Fusion Protein, in Patients with Malignant Melanoma or Renal Cell Carcinoma

PURPOSE: AS1409 is a fusion protein comprising a humanised antibody BC1 linked to interleukin-12 (IL-12). It is designed to deliver IL-12 to tumor-associated vasculature using an antibody targeting the ED-B variant of fibronectin. EXPERIMENTAL DESIGN: We conducted a phase 1 trial of weekly infusional AS1409 in renal carcinoma and malignant melanoma patients. Safety, efficacy, markers of IL-12-mediated immune response, and pharmacokinetics were evaluated. RESULTS: 11 melanoma and 2 renal cell carcinoma patients were treated. Doses of 15μg/kg and 25μg/kg were studied. Most drug-related adverse events were grade ≤2, and included pyrexia, fatigue, chills, headache, vomiting and transient liver function abnormalities. Three dose limiting toxicities of grade 3 fatigue and transaminase elevation were seen at 25μg/kg. IFN-γ and interferon-inducible protein-10 (IP-10) were elevated in all patients, indicating activation of cell-mediated immune response; this was attenuated at subsequent cycles. Anti-drug antibody responses were seen in all patients, although bioassays indicate these do not neutralise AS1409 activity. Plasma half-life was 22 hours and not dose-dependent. Five patients received ≥6 cycles and a best response of at least stable disease was seen in 6 (46%) patients. Partial response was seen in a melanoma patient, and disease shrinkage associated with metabolic response was maintained beyond 12 months in another melanoma patient despite previous rapid progression. CONCLUSIONS: The maximum tolerated dose was established at 15μg/kg weekly. AS1409 is well tolerated at this dose. Evidence of efficacy assessed by RECIST, functional imaging, and biomarker response warrants the planned further investigation using this dose and schedule in malignant melanoma