TPS8123 Background: D is an orally available, potent and selective irreversible small molecule inhibitor of all catalytically active members of the HER (human epidermal growth factor receptor) family tyrosine kinases that has shown activity in preclinical studies on EGFR mutant cell lines, including those resistant to G. In a phase II trial of NSCLC pts who received 1st-line D, 75.6% of 45 pts with confirmed EGFR exon 19 or 21 sensitizing mutations (m) experienced a partial response (PR). The median progression-free survival (PFS) was 18.2 mo, and the PFS rate at 1 yr was 76.5% (preliminary data; Mok et al APLCC 2012). Methods: Based on the phase II data, a phase III randomized, open label trial (ARCHER 1050; NCT01774721) was designed to compare the efficacy of 1st line D with G in pts with adv EGFR m-positive NSCLC. Eligible pts (N=440) have pathologically confirmed stage IIIB/IV NSCLC with at least one activating EGFR m, either exon 19 deletion or exon 21 L858R m. Concurrent m in exon 20 T790M is permitted. Pts must have radiologically measurable disease, ECOG PS 0–1 and no prior systemic therapy. Pts will be randomized (1:1) to receive D 45 mg or G 250 mg orally once daily. The primary endpoint is PFS by Independent Radiologic Review. Secondary endpoints include PFS by investigator assessment, overall survival (OS), OS at 30 mo, best overall response, duration of response, and safety and tolerability. Pt-reported outcomes (HRQoL and disease/treatment-related symptoms) were also assessed. Randomization will be stratified by race (Japanese vs mainland Chinese vs other East Asian vs nonEEast Asian), and EGFR m status (exon 19 deletion vs exon 21 L858R m). A minimum of 268 PFS events is required for 90% power to detect a PFS improvement of ≥50% in D vs G recipients using the intent-to-treat (ITT) analysis population (HR ≤0.667). A significant (0.025 significance level) 1-sided stratified log-rank test for PFS at the final PFS analysis will be indicative of a positive study outcome. An interim analysis is planned to assess safety and whether early discontinuation of the trial is required for futility. Clinical trial information: NCT01774721.

Extensive preclinical studies have validated nucleolin as a new therapeutic target in oncology. Confocal microscopy, immunohistochemistry, and cell fractionation studies have shown that this protein is highly overexpressed in the plasma membrane and cytoplasm of a wide variety of human hematological and solid tumor cells, but is usually undetectable on the cell surface or in the cytoplasm of the corresponding normal cells. We have utilized our licensed platform technology to generate a first-in-class panel of eight fully human monoclonal IgG1 antibodies (HuMAbs). These antibodies bind specifically to nucleolin on the tumor cell surface, which in turn elicits potent cytotoxicity to a variety of human tumor cell lines. One such example is CP101, which killed MV4-11 (AML), MCF-7 (breast), DU145 (prostate), PANC-1 and MIA PaCa-2 (pancreas) tumor cell lines with IC50 values ranging from (0.5-2.0 µg/ml; 3-12 nM) following exposure to the HuMAb for 96 hrs. These in vitro assays were performed in the absence of human complement and immune effector cells required for complement-dependent cellular cytotoxicity (CDCC) and antibody-dependent cellular cytotoxicity (ADCC), respectively. These results are consistent with published observations that anti-nucleolin HuMAbs can exploit the known shuttling function of cell surface nucleolin to gain intracellular access and induce direct tumor cytotoxicity. In contrast to its cytocidal effects on tumor cells, CP101 had no effect on the viability of either MCF-10A normal human breast epithelial cells or normal human CD19+ B cells, neither of which expresses nucleolin on the cell surface. In summary, the results suggest that anti-nucleolin HuMAbs are unique in that they can exert broad spectrum antitumor activity independently of the immune mechanism of CDCC and ADCC, while having no detectable effects on the viability of the corresponding normal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4623. doi:1538-7445.AM2012-4623

Amonafide-l-malate (amonafide) is a unique DNA intercalator that maintains activity in the presence of MDR mechanisms, a frequent cause of treatment-failure in secondary AML. 43 patients with relapsed/refractory or secondary AML or CML blast crisis were enrolled into two phase I dose-escalation studies investigating amonafide as monotherapy or in combination with cytarabine. 3/17 patients in the monotherapy trial and 10/26 patients in the combination trial achieved a complete remission. Between both trials responses occurred in 9/20 patients with secondary AML. Both trials demonstrated an acceptable safety profile and significant antileukemic activity in patients with poor-risk AML, especially those with secondary AML.

Amonafide (AS1413, amonafide L-malate) has shown activity in phase I and II clinical trials in patients with secondary AML (sAML) and is now in a pivotal phase III clinical trial in sAML. The drug\#8217;s clinical activity prompted further investigation of its mechanism of action. Our results showed that amonafide is a potent DNA topoisomerase II (Topo II)-active drug that differs from the classical Topo II inhibitors (daunorubicin, etoposide, mitoxantrone) in its reactivity towards Topo II-DNA complexes and because it is neither a substrate nor an inhibitor of P-glycoprotein. Specifically, amonafide intercalated into DNA and both inhibited the binding of human Topo II to DNA and interfered with ATP binding to Topo II in DNA decatentation assays. Amonafide also blocked the etoposide-induced cleavage of pRYG DNA. These data indicate that amonafide inhibits Topo II catalysis prior to the formation of Topo II-DNA cleavable complexes and suggest that amonafide induces less DNA damage than the classical Topo II inhibitors. In contrast to daunorubicin and etoposide, the IC 50 concentration of amonafide induced apoptosis in MV4-11 leukemia cells without inducing significant \#947;-histone-2AX phosphorylation, which occurs after the induction of DNA double-strand breaks. Furthermore, significant cell surface expression of NG2 (neuron-glial antigen 2), a biomarker associated with MLL gene fusions, was not observed in CEM cells treated with IC 50 concentrations of amonafide. When CEM or MV4-11 cells were incubated with concentrations of amonafide, etoposide, or daunorubicin that induced similar degrees of apoptosis, amonafide treatment resulted in only high molecular weight DNA fragmentation (50-300 kb), while etoposide and daunorubicin treatment resulted in extensive cleavage of DNA into fragments less than 50 kb. These results indicate that the mechanism of action of amonafide is distinct from the classical Topo II-active drugs. Our data are consistent with a chromatin disorganization model in which amonafide triggers apoptosis via inhibition of Topo II, which induces the release of single (50 kb) and multiple chromatin loops (>50 kb) from their attachment sites on the nuclear matrix. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1700.

Abstract 1047

13536 Background: Multidrug resistance (MDR) associated with Pgp overexpression in blasts is common in high-risk AML and is associated with poor outcomes. Erba et al (JCO 2007) reported a 42% complete remission (CR) rate in patients (pts) with sAML [prior MDS or treatment-related AML, (tAML)] (N = 88) treated with amonafide and std dose cytarabine. In laboratory studies of Pgp+ human leukemia cells, amonafide was neither a substrate nor an inhibitor of Pgp-mediated efflux, in contrast to daunorubicin (DNR). We sought to correlate the lack of Pgp effect with treatment outcome. Methods: AML blasts from 15 pts from the Phase 2 trial cited above were retrospectively assessed for Pgp expression and function as well as amonafide and DNR uptake and retention in the presence and absence of the Pgp inhibitor cyclosporin A. Pts: median age, 62 yrs (range 51–87); 7 with prior MDS; 8 had tAML; 10 with unfavorable cytogenetics. Pgp-mediated efflux was assessed by comparing uptake of the Pgp substrate DiOC2(3) in the presence and absence of the Pgp inhibitor PSC-833. Pgp- mediated transport (effluxapp) was calculated as differential uptake and retention of amonafide and DNR with (a) and without (b) PSC-833, normalized to apparent influx, using the formula [(a-b)/a]×100, reported as the mean±s.e.m. Results: The 15 sAML samples showed significantly less effluxapp of amonafide (5.2%±3.2) than of DNR (16%±2.1; p=0.0083). The unfavorable cytogenetic subset showed much less effluxapp of amonafide (0.13%±3.7) compared to DNR (16%±2.1; p=0.0015). CR pts also showed less effluxapp of amonafide (4.0%±6.7) than of DNR (21%±2.9; p=0.035). Conclusions: The relative lack of Pgp-mediated efflux of amonafide, compared to DNR, from sAML blasts provides a rationale for its observed clinical efficacy in the Phase 2 trial. Prospective assessment of these MDR parameters is underway in a randomized Phase 3 clinical trial in sAML comparing amonafide to DNR in combination with std dose cytarabine for remission induction. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Xanthus Pharmaceuticals Xanthus Pharmaceuticals Xanthus Pharmaceuticals

Background: Secondary acute myeloid leukemia (sAML), evolving from prior MDS or leukemogenic therapy (tAML), is associated with a poor prognosis. Many sAML patients are elderly with significant comorbidities. The incidence of unfavorable cytogenetics and expression of multidrug resistance (MDR) phenotype is higher in sAML blasts. Amonafide (AS1413) is a DNA intercalating agent and unique topoisomerase II (Topo II) inhibitor that, unlike the classical Topo II inhibitors (DNR, IDA, mitoxantrone, etoposide), does not affect the DNA/Topo II cleavable complex that results in DNA fragmentation. It does not act as either a substrate or an inhibitor of the MDR efflux pump, P-glycoprotein (Pgp), nor is it cross-resistant with classical Topo II inhibitors. A complete remission (CR) rate of 38.6%, with an additional 3.4% of patients achieving CRp, has previously been reported for this phase II trial. Updated findings from this trial including duration of remission data are reported here.

7027 Background: The poor prognosis for sAML (prior MDS or leukemogenic therapy, i.e., tAML) relates to disease (unfavorable cytogenetics and multidrug resistance (MDR) phenotype) and patient (pt) characteristics (elderly w/comorbid illnesses). Amonafide (Xanafide), a topoisomerase II inhibitor, is not a substrate or inhibitor of the MDR efflux pump, P-glycoprotein (Pgp) (Chau 2007 Leuk Res in press; O'Loughlin 2007 Blood 110:702a; Lundberg 2008 ASCO, submitted). A 42% CR rate in this Phase 2 trial was previously reported (Erba 2007 Proc Am Soc Clin Onc 25:373s). We report updated results & long-term follow up. Methods: sAML pts received amonafide 600 mg/m2 days 1–5 + ara-C 200 mg/m2 CIV days 1–7. A 2nd course could be given for persistent leukemia on day 14. CR pts received either stem cell transplant or intermediate/high dose ara-C depending on age. Centralized pathology review and DSMB were utilized. Primary endpoint was complete remission (CR) with or without (CRi) hematologic recovery. Median duration of follow-up is 208 days. Results: Enrollment 88 pts; median age 63 yrs (range 23–87); prior MDS, 45.5%; tAML 54.5%; unfavorable cytogenetics, 47%. Overall CR was 42%, 34 CR + 3 CRp (CR without recovery of platelets). CR rate was consistent across poor risk subgroups: age <60yrs, 39.4%; >60yrs, 43.6%. MDS → AML without and with prior therapy for MDS (mostly azacytidine), 43.5% & 36%; tAML, 40%; intermediate & unfavorable cytogenetics, 60% & 22%. 6 of 10 CR pts with informative cytogenetics achieved cytogenetic CR. 30 pts received post-remission therapy with ara-C (21), BMT (7), or both (2). Median duration of CR is >10 months, among age > 60 is > 9 months, with pts continuing on study. Kaplan-Meier estimate of continuous CR at 12 months is 43%, 47%, 64% and 57% for all pts, older pts (>60), pts with tAML, and pts with poor risk cytogenetics. Median overall survival (OS) is 7 months, and median OS for CR pts is >11 months. Safety profile was acceptable, death within 28 days was 20.5%. Conclusions: This study demonstrated robust and durable CR across poor-risk subsets of sAML, including older pts, tAML, and previously treated MDS → AML. Amonafide and ara-C may be a promising alternative for pts with sAML, especially those with over- expression of Pgp. This is being tested in a Phase 3 clinical trial. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Xanthus Pharmaceuticals Xanthus Pharmaceuticals Xanthus Pharmaceuticals

1055 Background: C1311 was designed from mitoxantrone to lessen cardiotoxicity. Although an inhibitor of topo II, its locus of action differs from other topo II inhibitors in that it does not affect the topo II/DNA cleavable complex. Phase I trial dose-limiting toxicity (DLT) was non-cumulative, transient neutropenia without significant nonhematologic toxicity including cardiac function despite prior anthracycline exposure (Isambert et al. Proc. ASCO 2006, Abst 2069). Methods: 53 breast cancer pts resistant to taxanes and anthracyclines and other agents. Median (range) of 3 prior regimens (range 2–6), including median (range) of 6 drugs (range 2–7). Most patients had visceral predominant disease (liver, lung). Initial dose from phase 1 was 480 mg/m2, 1 hr IV infusion on d 1, 8, and 15 of a 28 day cycle. The first 5 patients sustained gr 4 neutropenia (1/5 with febrile neutropenia); starting dose was reduced to 360 mg/m2/wk x 3 q28d for the subsequent 48 patients. Results: A total of 163 cycles of therapy were administered, for a total median cumulative dose of 3.9 gms/pt. Neutropenia was the only serious toxicity, overall 48%, Grade 3/4, 38% which was rapidly reversible within 7–10 days. One patient had thrombocytopenia, (nadir 62K, rapidly reversible). Other mild treatment-related Aes were anemia 14%, nausea 36%, vomiting 20%, and diarrhea 20% controlled with std supportive care. Serial ECGs showed no significant prolongation of the QTc interval, or abnormal T or U waves. Serial MUGA or ECHO compared to baseline showed a mean decrease of 6.7% in the LVEF following up to 9 cycles in 38 patients. The primary therapeutic objective was the overall RECIST response rate (CR+PR). An overall therapeutic effect was observed in 40% of patients. There were 2 PR lasting 6+ and 9+ months. 19 (36%) patients sustained stable disease (SD), with 10 pts with SD 4+ months including 2 patients continuing at 13+ and 14+ months. Conclusions: The safety profile for C1311 is manageable transient neutropenia without significant nonhematologic Aes including cardiac and other vital organ function. There was evident disease control in 40% of breast cancer refractory to taxanes, anthracyclines and multiple hormone and cytotoxic therapies. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Xanthus Pharmaceuticals Xanthus Pharmaceuticals Xanthus Pharmaceuticals Xanthus Pharmaceuticals

3238 Assessment of Pgp function in leukemic blasts provides useful prognostic information regarding rates and treatment response and may be used for therapy selection in acute myeloid leukemia (AML) patients. Traditional assessment of Pgp function has utilized fluorescent indicators, such as DiOC2(3)or rhodamine 123 (Rh123), as surrogates for drug uptake and efflux. The objectives of this study were 1) to compare the performance of these two commonly used probes and representative therapeutic drugs in bidirectional in vitro permeability models, and 2) to validate the methodology for measuring Pgp+ status in patient cells when exposed to actual drugs susceptible to MDR [eg, daunorubicin (DNR), epirubicin (EPI), etoposide (VP-16), idarubicin (IDA), and mitoxantrone (MIT)] versus the surrogate probes and amonafide, a topoisomerase II inhibitor, which is not an MDR substrate (O’Loughlin et al. Blood 2007). Pgp-mediated differential transport was assessed in Caco-2 cells (Pgplow) or MDR1-MDCK (Pgphigh) monolayer cells as a bidirectional efflux/influx ratio (BD) calculated from separate apical-to-basolateral (A-B) and basolateral-to-apical (B-A) drug permeability measurements or as a monodirectional efflux/influx ratio (MD) based on the change in B-A permeability, both performed before and after co-administration of cyclosporin A (CSA), an inhibitor of Pgp. Our results indicated a significant correlation between BD and MD. The two methods afforded the same rank order in extent of efflux for the cohort of test compounds in both Pgplow and Pgphigh. The net efflux of amonafide was

Over-expression of P-glycoprotein (Pgp+) has been related to resistance to classical Topo II inhibitors used in the treatment of AML and is common in patients with poor-prognosis, such as those with secondary AML (sAML). Since clinical trials with amonafide, a unique ATP-independent Topo II inhibitor, in combination with cytarabine, have shown significant efficacy for remission induction in patients with sAML, we compared the cytotoxic effect of amonafide (amonafide l-malate, Xanafide) to the classical Topo II inhibitors (daunorubicin, doxorubicin, idarubicin, etoposide, and mitoxantrone) in K562 leukemia cells and in the MDR subline, K562/DOX. Pgp expression was found to be approximately 6.5-fold greater in K562/DOX and causes the rapid efflux of these drugs from the leukemia cell. As a consequence, the LC(50) values for the classical Topo II inhibitor drugs tested were each increased up to 3 log units. A similar result was also observed in murine P388 and P388/ADR leukemia cells. Addition of cyclosporin A reversed K562/DOX resistance for the classical Topo II inhibitors, decreasing their LC(50) values to the levels observed with wild type cells but had no effect on amonafide potency in Pgp+ or wild type cells. Further examination of amonafide in bidirectional Caco-2 and MDR1-MDCK models confirmed that amonafide is neither a substrate nor inhibitor of Pgp. These observations suggest that amonafide is a promising therapeutic candidate directed toward bypassing this common mechanism of drug resistance encountered in the treatment of patients with AML, and possibly in other resistant hematological malignancies as well.

7065 Background: sAML portends a poor prognosis due to disease (unfavorable cytogenetics and multidrug resistance (MDR) phenotype) and patient (pt) characteristics (elderly w/comorbid illnesses). Amonafide, a topoisomerase II inhibitor, is not a substrate for the MDR efflux pump, P-glycoprotein (see Chau et al, ASCO 2007). Phase 1 trials of amonafide alone or together with ara-C reported a 50% CR rate (10/20) in pts with sAML (Allen et al, ASCO 2006). The dose-limiting toxicities of amonafide were manageable. Methods: In a Phase II trial pts with sAML (prior MDS or leukemogenic therapy, i.e., tAML) received amonafide 600 mg/m2/day on days 1–5 and CIV ara- C 200 mg/m2/day on days 1–7. A 2nd course could be given if day 14 marrow showed persistent leukemia. CR pts received either stem cell transplant or intermediate/high dose ara-C depending on age. An independent Data Safety Monitoring Board and central morphology review participated in the study. Primary endpoint was CR rate. Results: Enrollment has been completed. 44 % of 80 pts achieved CR. Details are available for the 1st 40 pts: median age 63 yrs (range 26 –87); prior MDS, 57%; tAML, 43%; unfavorable cytogenetics, 40%. 16/40 (40%) achieved CR (15 CR+1CRp). Subgroup CR analysis: MDS→AML without and with prior therapy for MDS (mostly azacytidine), 64% & 33%; tAML, 29%; intermediate and unfavorable cytogenetics, 58% and 18%; <60yrs, 42%; ≥60yrs, 39%. With a median follow up of 39 weeks, the median duration of CR is 28 weeks (range 20+ to 40+ weeks), with 8 of 16 patients in continuous CR. Median overall survival for CR patients has not been reached (range 1–47+ weeks). Death in first 28 days from consequences of hypoplasia was 17.5%. Grade 3, 4 non-heme adverse events occurring in > 10% pts were hypotension 23%, pneumonia 18%, dyspnea 13%, and diarrhea 13%. Data on all patients will be updated. Conclusions: Amonafide and ara-C is well tolerated in patients with poor prognosis sAML. A 64% CR was achieved in pts with untreated MDS→AML. Lower CR occurred with prior therapy for MDS, tAML and unfavorable cytogenetics. Age ≥60 yrs did not affect CR. Amonafide and ara-C may be a promising alternative for patients with sAML, especially those with over-expression of P-glycoprotein. No significant financial relationships to disclose.

6584 Background: Patients (pts) with secondary AML (AML following an antecedent hematologic disorder or following known exposure to leukemogenic agents) are often elderly, with comorbid illness, and have a dismal prognosis. Secondary AML responds poorly to standard chemotherapy, with low response rates and short durations of remission. Amonafide (Am) is an ATP independent topoisomerase 2 inhibitor that in prior clinical studies had myelosuppression as the dose-limiting toxicity. Two phase 1 trials evaluated Am as either a single agent or in combination with ara-C in pts with relapsed/refractory or secondary AML. This retrospective review was performed to assess the activity of Am specifically in pts with secondary AML. Methods: In study AM-03, 5 pts with secondary AML were treated with a fixed dose (1000, 1100, or 1400 mg/m2/day) of Am IV over 2 hrs daily × 5. In study AM-04, 15 pts with secondary AML were treated with ara-C 200 mg/m2/day CIV days 1–7 and a fixed dose (600, 700 or 800 mg/m2/d) of Am IV over 2 hrs daily on days 1–5. All clinical trial data were reviewed for assessment of hematological and cytogenetic response. Results: Across the two studies, 10 of 20 (50%) pts responded to Am (9 CR, 1 CRi). In study AM-03 (Am monotherapy), 3 of 5 (60%) pts responded, with median duration of response 7.5 months (range 2.5 - 8). None of these 3 pts received additional post-remission therapy. In study AM-04 (Am + ara-C), 7 of 15 pts (47%) responded, with median duration of response 8 months (range 1 ¾ to >60 months). 4 of these 7 pts received additional post-remission therapy. Cytogenetic analysis prior to and following Am therapy was available for 9 of the 10 pts with secondary AML who responded to Am in the 2 studies. Cytogenetics were abnormal prior to Am therapy in 5 of these 9 pts, 3 (60%) of whom achieved cytogenetic CR. Conclusions: Am shows significant activity in secondary AML, both as monotherapy and in combination with ara-C. A phase 2 trial of Am + ara-C in pts with secondary AML is currently enrolling patients. [Table: see text]

Planning therapy for acute myelogenous leukemia (AML) is difficult because of the heterogeneous nature of the disease and varying patient age at presentation. Cytogenetics and patient age at the time of diagnosis are two major factors determining treatment outcome in AML. Patients with poor-risk cytogenetics have much lower complete remission rates than other groups. In addition, AML in patients greater than 55 to 60 years of age often exhibits a resistant phenotype, more akin to secondary AML or AML arising from myelodysplastic syndromes. This group is also characterized by lower complete remission rates, and often requires the delivery of intensive therapy to a patient population that is the least likely to tolerate it. At the Jefferson Health System (Philadelphia, PA), we wished to develop a regimen that was maximally intensive to treat stubborn disease, but gentle enough to be given to all patients regardless of age. Toward this end, 33 patients received a maximal dose of the cytoprotective agent, amifostine, before each infusion of idarubicin in the "7 + 3" regimen, escalating the dose of idarubicin in a phase I fashion to a maximum dose of 24 mg/m2 . The data indicate that the addition of amifostine to "7 + 3" AML induction therapy enables a substantial escalation of the idarubicin dose through the 21-mg/m2 dose level, without a concomitant increase in side effects, thus providing a regimen that is both intensive and applicable to patients of all ages. Currently, phase II studies are ongoing on a national basis to evaluate the efficacy of this regimen.

An ideal treatment for lymphoma and leukemia is the use of highly selective compounds to eliminate diseased cells with minimal systemic toxicity to normal tissues (cf. imatinib mesylate; Gleevec). AQ4N (1,4 bis[[2-(dimethylamino)ethylamino}-5,8-hydroxyanthracene-9,10-dione bis N-oxide) is designed to have little or no toxicity until selectively activated by bioreduction in hypoxic cells to AQ4 (reduced AQ4N), a highly potent DNA topoisomerase II inhibitor. In a series of studies, AQ4 has been shown to have potent cytotoxicity on lymphoma and leukemia cell lines in vitro and AQ4N has selective activity in lymphatic tissues in vivo. The IC50 of AQ4, was 0.63, 12.0, 90.5 and 150 nM in Namalwa, Daudi, Ramos, and Raji human lymphoma cell lines and 1.0, 6.0, and 20 nM in HL-60, KG1a and K562 human leukemia cell lines. On several of the tumor lines the activity of AQ4 was more potent than doxorubicin (i.e. IC50 for Dox was 20.3 nM on Namalwa). AQ4N also had anti-proliferative activity at μM levels indicating a potential mechanism for activation by these cell lines. In repeat dose toxicology studies of AQ4N in pigmented rats and cynomolgus monkeys, the maximum tolerated doses (MTD; rats: 20 mg/kg/wk x 6; monkeys 6 mg/kg/wk x 6) resulted in lymphoid tissue atrophy. A decrease in lymphocyte levels and atrophy of the spleen, thymus, and mandibular and mesenteric lymph nodes were observed at terminal sacrifice of the animals. In contrast, there was an absence of myelosuppression and only mild neutropenia and minor bone marrow atrophy at the MTD. Administration of radiolabeled AQ4N (14C-benzene) to pigmented rats and cynomolgus monkeys indicated persistence of AQ4N radioactivity in lymphoid tissues for several weeks after a single dose (rats: 20 mg/kg (130–140 μCi/kg); monkeys: 10 mg/kg (135 μCi/kg)). For example, in rats the half-life of radioactive AQ4N in the spleen was 538 hrs with 0.9 μg AQ4N/g tissue (spleen) remaining one week after dosing. Monkeys demonstrated a similar effect with 76.5–86.8 μg AQ4N/g tissue observed in the spleen one week after treatment. Other tissues contained significantly less radioactive AQ4N with the exception of the liver (67.9–78.6 μg AQ4N/g tissue) and adrenal cortex (78.7–86.6 μg AQ4N/g tissue). While some hypertrophy and eosinophila was observed in the adrenal glands, liver toxicity was not observed at the MTD in the repeat dose cynomolgus monkey toxicology study. Overall, these initial findings indicate that AQ4N is active in vitro against human lymphoma and leukemia cell lines and selectively targets lymphoid tissues in vivo suggesting the potential benefit of AQ4N in the treatment of lymphoproliferative diseases.

Patients with low grade (LG) non-Hodgkin's lymphomas (NHLs) typically have a median survival of 8-10 years during which they sustain a series of responses and relapses to therapy. More than 95% of B-cell NHLs express the CD20 surface antigen, affording opportunities for CD20-directed therapy of NHL. Since 1990, 5 trials have tested the safety and efficacy of the murine CD20 MAb Tositumomab and Iodine I 131 Tositumomab (Bexxar therapeutic regimen) in 250 patients with relapsed, refractory, or transformed LG NHL. The I-131 irradiates MAb-bound cells and those within the path length of the isotope. Response rates were 56% (overall) and 30% (complete). With a median follow-up of 44.6 months, 30% of the patients achieved a long-term, durable response; median time to progressive disease or death was 5 years. Thus, a single treatment with Bexxar may produce durable responses and partially reverse the natural history of LG NHL.

The objectives of this study were to evaluate the protective effects of amifostine against paclitaxel-induced toxicity to normal and malignant human tissues. Haematopoietic progenitor colony assays were used to establish the number of CFU-GEMM and BFU-E colonies after incubation with WR-1065 alone, Amifostine alone, paclitaxel (2.5 or 5 μM) +/- WR-1065 or amifostine. MTT and alkaline elution assays evaluated the in vitro growth inhibitory and DNA damaging effects, respectively, of paclitaxel with or without amifostine against normal human fibroblasts and human non-small cell lung cancer (NSCLC) cells. This combination was also evaluated in vivo using severe combined immune deficient (scid) mouse models of early (non-palpable tumours) and advanced (palpable tumours) human ovarian cancer. Human 2780 ovarian cancer cells were inoculated subcutaneously while paclitaxel and amifostine were administered intraperitoneally. A brief exposure (15 min) to amifostine not only protected human haematopoietic progenitor colonies from paclitaxel toxicity, but stimulated the growth of CFU-GEMM and BFU-E beyond control values. Amifostine protected normal human lung fibroblasts from paclitaxel-induced cytotoxicity and DNA single-strand breaks. However, paclitaxel cytotoxicity and DNA single-strand breaks were actually enhanced by pretreatment with amifostine in the NSCLC model. Importantly, amifostine did not interfere with paclitaxel antitumour activity even with prolonged exposure (24.5 h) of the lung cancer cells to high concentrations (1.2 mM) in vitro or following five repetitive high doses (200 mg/kg) given to scid mice with human ovarian cancer xenografts. Indeed, under certain circumstances, amifostine resulted in sensitisation of tumour cells to paclitaxel. Our results confirm previous reports of the ability of amifostine to protect normal tissues from the toxic effects of chemotherapy drugs and now extend these observations to paclitaxel.