SG2000
Puzanov, I., Lee, W., Chen, A.P., Calcutt, M.W., Hachey, D.L., Vermeulen, W.L., Spanswick, V.J., Liao, C-Y., Hartley, J.A., Berlin, J.D., and Rothenberg, M.L.: Phase I, pharmacokinetic and pharmacodynamic study of SJG-136, a novel DNA sequence selective minor groove cross-linking agent, in advanced solid tumors. Clinical Cancer Res. 2011, 17, 3794-802,
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PURPOSE:
This phase I study assessed the maximum tolerated dose (MTD), safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of SJG-136, a sequence-specific DNA cross-linking agent, in patients with advanced cancer.
EXPERIMENTAL DESIGN:
In schedule A, seven patients received escalating doses of SJG-136 (6, 12, 24, and 48 μg/m(2)) daily for 5 of 21 days. Blood samples were collected for PK analysis on days 1 and 5 of cycle 1. In schedule B, SJG-136 was given daily for 3 of 21 days (N = 17; doses 20, 25, 30, and 35 μg/m(2)). Blood samples were collected on days 1 and 3 of cycles 1 and 2 for PK and PD analysis. Patients in schedule B received dexamethasone and early diuretic care.
RESULTS:
Schedule A-dose-limiting toxicities included grade 3 edema, dyspnea, fatigue, and delayed liver toxicity (grade 3-4). PK analysis revealed dose-dependent increases in AUC and C(max). Substantial changes in volume of distribution at steady-state occurred after repeated dosing in some patients prior to the onset of edema. Schedule B-the same toxicities were manageable with steroid premedication and diuretic support. No significant myelosuppression occurred on either schedule. DNA interstrand cross-links correlated with systemic exposure of SJG-136 following the second dose in cycle 1 and were still detectable immediately before cycle 2.
CONCLUSIONS:
The MTD of SJG-136 in this study was 30 μg/m(2) administered on a daily 3× basis with no myelosuppression effects. Coupled with supportive management, SJG-136 is now advancing to a phase II trial in ovarian cancer.
Janjigian, Y.; Lee, W.; Kris, M.; Miller, V.; Krug, L.; Azzoli, C.; Senturk, E.; Wade Calcutt, M.; Rizvi, N. A phase I trial of SJG-136 (NSC#694501) in advanced solid tumors. Cancer Chemotherapy and Pharmacology 2010, 65, 833-83
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Purpose: SJG-136 is a pyrrolobenzodiazepine dimer that forms DNA crosslinks and has demonstrated broad antitumor activity. We undertook this trial to determine the maximum-tolerated dose (MTD), toxicities and pharmacokinetic (PK) profile of SJG-136 in patients with an advanced solid tumor.
Patients and methods: In this phase I study, patients were treated with SJG-136 on days 1, 8 and 15 of a 28-day cycle. Dose levels studied were 10, 20, 40 and 60 μg/m2. PK parameters of SJG-136 were assessed following the intravenous administration of SJG-136 on days 1 and 15 of cycle 1.
Results: Twenty-one patients with advanced solid tumors were treated. Patients had a median of two prior chemotherapy regimens. Fatigue was dose-limiting with SJG-136 60 μg/m2/day administered on days 1, 8 and 15 of a 28-day cycle. Grade 3 thrombocytopenia and delayed onset liver toxicity were seen in one patient each. PK parameters of SJG-136 indicated dose-proportional increases in systemic exposure with increasing doses. No objective responses were seen.
Conclusion: For patients with advanced solid tumors, the MTD of SJG-136 is 40 μg/m2/day administered on days 1, 8 and 15 of a 28-day cycle. The major dose limiting toxicity was fatigue. Alternative dosing strategies are now being evaluated.
Hochhauser, D.; Meyer, T.; Spanswick, V. J.; Wu, J.; Clingen, P. H.; Loadman, P. M.; Cobb, M.; Gumbrell, L.; Begent, R. H. J.; Hartley, J. A.; Jodrell, D. I. Phase I Study of Sequence-Selective Minor Groove DNA Binding Agent SJG-136 in Patients with Advanced Solid Tumors. Clinical Cancer Research 2009, 15, 2140-2147
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Purpose: This phase I dose-escalation study was undertaken to establish the maximum tolerated dose of the sequence-selective minor groove DNA binding agent SJG-136 in patients with advanced solid tumors. The study also investigated antitumor activity and provided pharmacokinetic and pharmacodynamic data. Experimental design: Sixteen patients were assigned sequentially to escalating doses of SJG-136 (15-240 microg/m(2)) given as a 10-minute i.v. infusion every 21 days. The dose was subsequently reduced in incremental steps to 45 microg/m(2) due to unexpected toxicity. Results: The maximum tolerated dose of SJG-136 was 45 microg/m(2). The main drug-related adverse event was vascular leak syndrome (VLS) characterized by hypoalbuminemia, pleural effusions, ascites, and peripheral edema. Other unexpected adverse events included elevated liver function tests and fatigue. The VLS and liver toxicity had delayed onset and increased in severity with subsequent cycles. Disease stabilization was achieved for >6 weeks in 10 patients; in 2 patients this was maintained for >12 weeks. There was no evidence of DNA interstrand cross-linking in human blood lymphocytes with the use of the comet assay. Evidence of DNA interaction in lymphocytes and tumor cells was shown through a sensitive gamma-H2AX assay. SJG-136 had linear pharmacokinetics across the dose range tested. Conclusions: SJG-136 was associated with dose-limiting VLS and hepatotoxicity when administered by short injection every 21 days. DNA damage was noted, at all dose levels studied, in circulating lymphocytes. The etiology of the observed toxicities is unclear and is the subject of further preclinical research. Alternative clinical dosing strategies are being evaluated.
Rahman, K. M.; Thompson, A. S.; James, C. H.; Narayanaswamy, M.; Thurston, D. E. The Pyrrolobenzodiazepine Dimer SJG-136 Forms Sequence-Dependent Intrastrand DNA Cross-Links and Monoalkylated Adducts in Addition to Interstrand Cross-Links. Journal of the American Chemical Society 2009, 131, 13756-13766
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SJG-136 (1) is a sequence-selective DNA-interactive agent that is about to enter phase II clinical trials. Using a HPLC/MS-based methodology developed to evaluate the binding of DNA-interactive agents to oligonucleotides of varying length and sequence, we have demonstrated that, in addition to the previously known interstrand cross-link at Pu-GATC-Py sequences, 1 can form a longer interstrand cross-link at Pu-GAATC-Py sequences, an intrastrand cross-link at both shorter Pu-GATG-Py and longer Pu-GAATG-Py sequences, and, in addition, monoalkylated adducts at suitable PBD binding sites where neither intra- or interstrand cross-links are feasible because of the unavailability of two appropriately positioned guanines. Crucially, we have demonstrated a preference for the extended intrastrand cross-link with Pu-GAATG-Py, which forms more rapidly than the other cross-links (rank order: Pu-GAATG-Py > Pu-GATC-Py >> Pu-GATG-Py and Pu-GAATC-Py). However, thermal denaturation studies suggest that the originally reported Pu-GATC-Py interstrand cross-link is more stable, consistent with the covalent joining of both strands of the duplex and a lower overall distortion of the helix according to modeling studies. These observations impact on the proposed mechanism of action of SJG-136 (1) both in vitro and in vivo, the repair of its adducts and mechanism of resistance in cells, and potentially on the type of pharmacodynamic assay used in clinical trials.
Narayanaswamy, M.; Griffiths, W. J.; Howard, P. W.; Thurston, D. E. An Assay Combining High-Performance Liquid Chromatography and Mass Spectrometry to Measure DNA Interstrand Cross-Linking Efficiency in Oligonucleotides of Varying Sequences. Analytical Biochemistry 2008, 374, 173-181
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The main method of evaluating the DNA interstrand cross-linking ability of cancer chemotherapeutic agents in naked DNA currently involves the electrophoresis of relatively long radiolabeled duplex DNA fragments (typically approximately 2000 bp) on neutral gels after incubation with the agent of interest. Denaturation by heating is carried out prior to loading, and a neutral gel allows reannealing of cross-linked DNA. To avoid the use of radioactivity we have developed a new method based on ion pair reversed phase liquid chromatography (RPLC) and mass spectrometry (MS) that allows characterization and quantitation of drug-DNA interstrand cross-links formed within short oligonucleotide duplexes (i.e., 12 bp). Advantages of this assay include rapid throughput, as compared to electrophoretic methods, and the use of readily available short nonradiolabeled oligonucleotides of any sequence, thereby facilitating investigation of sequence selectivity. A further advantage is that all species separated by the chromatographic process can be positively identified by MS. Using this new method, we have investigated the rate of DNA cross-linking and sequence selectivity of the interstrand cross-linking agent SJG-136, a pyrrolobenzodiazepine (PBD) dimer currently in phase I clinical trials. The assay was found to be sufficiently sensitive and selective to allow separation of the unbound and drug-bound oligonucleotide species by high-performance liquid chromatography (HPLC) and to allow positive identification of these individual species by MS. A further benefit, as compared with electrophoretic methods, is that kinetic information can be obtained and compared for different binding sequences.
Wade, C. M.; Lee, W.; Puzanov, I.; Rothenberg, M. L.; Hachey, D. L. Determination of chemically reduced pyrrolobenzodiazepine SJG-136 in human plasma by HPLC-MS/MS: application to an anticancer phase I dose escalation study. Journal of Mass Spectrometry 2008, 43, 42-52.
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SJG-136 1,1'-[[(propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-methylidene-1,2,3,11a-tetrahydro-5H-pyr- rolo[2,1-c][1,4]benzodiazepin-5-one]] (NSC 694501), is a bifunctional pyrrolobenzodiazepine (PBD) dimer that forms selective, irreversible, interstrand DNA cross-links via exocyclic N2 atoms of two guanine bases, with a preference for 5'PuGATCPy binding sites. SJG-136 is highly cytotoxic in human tumor cells in vitro and in human tumor xenograft models in vivo at subnanomolar concentrations and is currently in anticancer phase I clinical trials in the United Kingdom and United States. To support correlative pharmacokinetics studies, a highly sensitive HPLC-MS/MS assay was developed and validated for the reliable quantitation of SJG-136 in human plasma, using the structurally similar PBD dimer DSB-120 as an internal standard. Chemical reduction of SJG-136 to its corresponding amine (SJG-136-H(4), [M + H](+)m/z 561) improved HPLC peak resolution and sensitivity by minimizing complications that arose from the reactivity of the labile imine moieties. Plasma samples were processed by protein precipitation and centrifugal membrane dialysis; components were separated by HPLC using an Agilent Rapid Resolution HT 1.8 mm (2.1 mm x 50 mm) analytical column. The total analysis time from injection to injection was 11 min. Electrospray MS/MS detection of SJG-136-H(4) was based on the selected reaction monitoring (SRM) transition [M + H](+)m/z 561 --> 301. The analytical response ratio was linearly proportional to the plasma concentration of SJG-136 over the nominal concentration range of 25 pg/ml to 250 ng/ml, with a coefficient of determination of r > or = 0.999. The intrarun absolute %RE was < or =19.6, 14.2, and 14.0% at 0.056, 2.83, and 56.3 ng/ml, respectively. The corresponding %RSD was < or =14.9%, 9.01, and 4.59%. The interday %RSD was < or =2.72, 3.46, and 5.20%. The lower and upper limits of quantitation were 0.056 and 56 ng/ml, respectively; recovery of SJG-136 from plasma was > or = 62% across the validated concentration range. The sensitivity of the validated assay was sufficient to detect SJG-136 in human subjects for up to 6 h after intravenous administration of 6 microg/m(2), the starting dose of an NCI-sponsored dose escalation study.
Aird, R. E.; Thomson, M.; Macpherson, J. S.; Thurston, D. E.; Jodrell, D. I.; Guichard, S. M. ABCB1 genetic polymorphism influences the pharmacology of the new pyrrolobenzodiazepine derivative SJG-136. Pharmacogenomics Journal 2008, 8, 289-296
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ATP-binding cassette transporter P-glycoprotein (ABCB1) is responsible for the multidrug resistance (MDR1) phenotype observed in cancer cells. SJG-136, a new pyrrolobenzodiazepine dimer, is a sequence-dependent DNA crosslinking agent and substrate of ABCB1. We previously showed that colon cancer cell lines expressing high levels of ABCB1 showed a lower sensitivity to SJG-136. Here, we show that in 3T3 isogenic fibroblasts, ABCB1 genetic polymorphism differentially affects ABCB1 gene expression and transport function. However, this genotype-phenotype relationship was not observed in immortalized lymphocytes, which expressed 10- to 1000-fold less ABCB1 than colon cancer cell lines. Consistent with this, the cytotoxicity of SJG-136 in 3T3 fibroblasts was affected by ABCB1 genetic polymorphism but not in immortalized lymphocytes. ABCB1 genetic polymorphism is therefore likely to affect drug sensitivity in tissues expressing high levels of the transporter and in which significant variability is observed.
Pepper, C.; Lowe, H.; Fegan, C.; Thurieau, C.; Thurston, D. E.; Hartley, J. A.; Delavault, P. Fludarabine-Mediated Suppression of the Excision Repair Enzyme ERCC1 Contributes to the Cytotoxic Synergy with the DNA Minor Groove Crosslinking Agent SJG-136 (NSC 694501) in Chronic Lymphocytic Leukaemia Cells. British Journal of Cancer 2007, 97, 253-259.
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In this study, we set out to establish whether fludarabine could enhance the DNA interstrand crosslinking capacity of SJG-136 in primary human chronic lymphocytic leukaemia (CLL) cells and thereby offer a rationale for its clinical use in combination with SJG-136. SJG-136 rapidly induced DNA crosslinking in primary CLL cells which was concentration-dependent. Further, the level of crosslinking correlated with sensitivity to SJG-136-induced apoptosis (P=0.001) and higher levels of crosslinking were induced by the combination of SJG-136 and fludarabine (P=0.002). All of the samples tested (n=40) demonstrated synergy between SJG-136 and fludarabine (mean combination index (CI)=0.54+/-0.2) and this was even retained in samples derived from patients with fludarabine resistance (mean CI=0.62+/-0.3). Transcription of the excision repair enzyme, ERCC1, was consistently increased (20/20) in response to SJG-136 (P<0.0001). In contrast, fludarabine suppressed ERCC1 transcription (P=0.04) and inhibited SJG-136-induced ERCC1 transcription when used in combination (P=0.001). Importantly, the ability of fludarabine to suppress ERCC1 transcription correlated with the degree of synergy observed between SJG-136 and fludarabine (r(2)=0.28; P=0.017) offering a mechanistic rationale for the synergistic interaction. The data presented here provides a clear indication that this combination of drugs may have clinical utility as salvage therapy in drug-resistant CLL.
Gallmeier, E.; Calhoun, E. S.; Rago, C.; Brody, J. R.; Cunningham, S. C.; Hucl, T.; Gorospe, M.; Kohli, M.; Lengauer, C.; Kern, S. E. Targeted Disruption of FANCC and FANCG in Human Cancer Provides a Preclinical Model for Specific Therapeutic Options. Gastroenterology 2006, 130, 2145-2154.
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Background and aims: How specifically to treat pancreatic and other cancers harboring Fanconi anemia gene mutations has raised great interest recently, yet preclinical studies have been hampered by the lack of well-controlled human cancer models.
Methods: We endogenously disrupted FANCC and FANCG in a human adenocarcinoma cell line and determined the impact of these genes on drug sensitivity, irradiation sensitivity, and genome maintenance.
Results: FANCC and FANCG disruption abrogated FANCD2 monoubiquitination, confirming an impaired Fanconi anemia pathway function. On treatment with DNA interstrand-cross-linking agents, FANCC and FANCG disruption caused increased clastogenic damage, G2/M arrest, and decreased proliferation. The extent of hypersensitivity varied among agents, with ratios of inhibitory concentration 50% ranging from 2-fold for oxaliplatin to 14-fold for melphalan, a drug infrequently used in solid tumors. No hypersensitivity was observed on gemcitabine, etoposide, 3-aminobenzamide, NU1025, or hydrogen peroxide. FANCC and FANCG disruption also resulted in increased clastogenic damage on irradiation, but only FANCG disruption caused a subsequent decrease in relative survival. Finally, FANCC and FANCG disruption increased spontaneous chromosomal breakage, supporting the role of these genes in genome maintenance and likely explaining why they are mutated in sporadic cancer.
Conclusions: Our human cancer cell model provides optimal controls to elucidate fundamental biologic features of individual Fanconi anemia gene defects and facilitates preclinical studies of therapeutic options. The impact of Fanconi gene defects on drug and irradiation sensitivity renders these genes promising targets for a specific, genotype-based therapy for individual cancer patients, providing a strong rationale for clinical trials.
Buhrow, S. A.; Reid, J. M.; Jia, L.; McGovern, R. M.; Covey, J. M.; Kobs, D. J.; Grossi, I. M.; Ames, M. M. LC-MS/MS assay and dog pharmacokinetics of the dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501). Journal of Chromatography B 2006, 840, 56-62.
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The dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501) has potent in vitro cytotoxicity and in vivo antitumor activity. SJG-136 binds in the minor groove of DNA and produces G-G interstrand cross-links via reactive N(10)-C(11)/N(10')-C(ll') imine/carbinolamine moieties. We have developed a sensitive, specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method for the quantitative determination of SJG-136 in plasma. SJG-136 was isolated by solid phase extraction through a C8 column, reverse-phase HPLC separation was accomplished on a C18 column with isocratic elution and MS/MS detection, monitoring the m/z 557-m/z 476 transition after electrospray ionization. The linear range and lower limit of quantitation from plasma standard curves were 2.8-1800 nM, and 5 nM, respectively. SJG-136 plasma protein binding was species-dependent. Values of the unbound fraction in human, rat and mouse were 25%, 16.2% and <1%, respectively. Protein binding was saturable in dog plasma where the unbound fraction increased from 10.8% to 22.3% over a 22-720 nM concentration range. SJG-136 pharmacokinetics after a single intravenous dose were best fit to a two-compartment open model with elimination half-life and plasma clearance values of 97 min and 6.1 mL/min/kg, respectively. SJG-136 did not accumulate in plasma following intravenous administration of 1.0 microg/kg doses for five consecutive days.