Phase II Trial of Infusional Fluorouracil, Irinotecan, and Bevacizumab for Metastatic Colorectal Cancer: Efficacy and Circulating Angiogenic Biomarkers Associated With Therapeutic Resistance

Purpose We investigated the efficacy of fluorouracil (FU), leucovorin, irinotecan, and bevacizumab (FOLFIRI + B) in a phase II trial in patients previously untreated for metastatic colorectal cancer (mCRC), and changes during treatment in plasma cytokines and angiogenic factors (CAFs) as potential markers of treatment response and therapeutic resistance. Patients and Methods We conducted a phase II, two-institution trial of FOLFIRI + B. Each 14-day cycle consisted of bevacizumab (5 mg/kg), irinotecan (180 mg/m(2)), bolus FU (400 mg/m(2)), and leucovorin (400 mg/m(2)) followed by a 46-hour infusion of FU (2,400 mg/m(2)). Levels of 37 CAFs were assessed using multiplex-bead assays and enzyme-linked immunosorbent assay at baseline, during treatment, and at the time of progressive disease (PD). Results Forty-three patients were enrolled. Median progression-free survival (PFS), the primary end point of the study, was 12.8 months. Median overall survival was 31.3 months, with a response rate of 65%. Elevated interleukin-8 at baseline was associated with a shorter PFS (11 v 15.1 months, P = .03). Before the radiographic development of PD, several CAFs associated with angiogenesis and myeloid recruitment increased compared to baseline, including basic fibroblast growth factor (P = .046), hepatocyte growth factor (P = .046), placental growth factor (P < .001), stromal-derived factor-1 (P = .04), and macrophage chemoattractant protein-3 (P < .001). Conclusion Efficacy and tolerability of FOLFIRI + B appeared favorable to historical controls in this single arm study. Before radiographic progression, there was a shift in balance of CAFs, with a rise in alternate pro-angiogenic cytokines and myeloid recruitment factors in subsets of patients that may represent mechanisms of resistance.

Photodegradation of irinotecan (CPT-11) in aqueous solutions: Identification of fluorescent products and influence of solution composition

The photodegradation of irinotecan (CPT-11), the semisynthetic derivative of the antitumor alkaloid 20(S)-camptothecin, has been investigated. The drug was exposed to laboratory light for up to 5 days in 0.9% saline solution (pH 8.5). Five significant photodegradation products were observed and a high-performance liquid chromatography (HPLC) assay was employed to isolate them from CPT-11 using gradient conditions. The structures were elucidated by nuclear magnetic resonance spectroscopy and tandem mass spectrometry and shown to be the result of extensive modifications of the lactone ring of CPT-11. Three of the compounds were found to belong to the mappicine group of alkaloids. In addition, the effect of light on the stability of CPT-11 in aqueous solutions and biological fluids was also assessed, Potassium phosphate buffers (0.05 M, pH 5.0-8.2) and saline, plasma, urine, and bile solutions containing 20 mu M CPT-11 were equilibrated in the dark for 24 h before being exposed to laboratory light for up to 171 h at ambient temperature. Four of the five identified photodegradation products were observed and quantitated by isocratic HPLC, using a different detection mode (fluorescence) than the one used for gradient elution, In general, CPT-11 was found to be unstable under neutral and alkaline conditions for all solutions investigated, with the exception of bile. We conclude that CPT-11 is photolabile and that care should be taken to protect samples, particularly those intended for the isolation and identification of novel metabolites of CPT-11.

Irinotecan-induced central nervous system toxicity: a case report.

Here we describe the first case, to our knowledge, of CNS [Central nervous system] toxicity in a patient treated with irinotecan.

Effectiveness of the bevacizumab-irinotecan regimen in the treatment of recurrent glioblastoma multiforme: Comparison with other second-line treatments without this regimen.

A retrospective cohort study was conducted to analyse the effectiveness of bevacizumab and irinotecan (BVZ/CPT-11) as a second-line treatment in patients with primary glioblastoma multiforme (GBM) in comparison with a control group that were not administered BVZ/CPT-11 at the first recurrence. The difference in overall survival (OS) between the two groups was used as a predictor of effectiveness. OS was calculated according to prognostic factors and gender. A total of 28 and 32 patients were enrolled in the BVZ/CPT-11 cohort and control group, respectively. The median OS was 17.94 months (95% CI, 14.91-20.96) in the BVZ/CPT-11 treatment cohort and 10.97 months (95% CI, 7.65-14.30) in the control cohort. The results obtained on the effectiveness of BVZ/CPT-11 treatment in patients with primary GBM are consistent with data from previous studies. No significant differences were identified in OS based on prognostic factors; therefore, the latter cannot be used to select patients who would incur the greatest benefits from BVZ/CPT-11 treatment.

Irinotecan loaded in eluting beads: preclinical assessment in a rabbit VX2 liver tumor model.

PURPOSE: The purpose of this study was to study the pharmacokinetics of irinotecan injected intravenously, intra-arterially, or loaded onto a delivery platform. MATERIAL AND METHODS: Fifty-four New Zealand White rabbits with VX2 liver tumor, divided in 3 groups of 17 rabbits, each received irinotecan either by intravenous (IV) route, intra-arterial hepatic (IA) route, or loaded on drug-eluting beads (DEBIRI). Animals were killed at 1, 6, and 24 h. Irinotecan and SN-38 concentrations were measured at different time points in serum, tumor, and normal liver. RESULTS: Twelve milligrams of irinotecan were injected IV and IA, whereas 6-16.5 mg were injected loaded onto DEBIRI. Normalized serum irinotecan reached a peak of 333 ng/ml (range 198.8-502.5) for IV, 327.1 ng/ml (range 277.1-495.6) for IA, and 189.7 ng/ml (range 111.1-261.9) for DEBIRI (P < 0.001) delivery. The area-under-the-curve value from 10 to 60 min of serum irinotecan concentration was significantly lower for DEBIRI (P = 0.0009). Tumor irinotecan levels for IV, IA, and DEBIRI (in ng/200 mg of tissue followed by ranges in parentheses) were, respectively, 23.6 (0.3-24.9), 36.5 (7.7-1914.1), and 20.2 (2.9-319) at 1 h; 4.2 (1-27.9), 99.3 (46.6-159.5), and 42.1 (11.3-189) at 6 h; and 2.7 (2.5-6.9), 18.3 (1.5-369.1), and 174.4 (3.4-5147.3) at 24 h (P = 0.02). At 24 h, tumor necrosis was 25% (10-30), 60% (40-91.25), and 95% (76.25-95) for IV, IA, and DEBIRI, respectively (P = 0.03). CONCLUSION: Compared with IV or IA, DEBIRI induces lower early serum levels of irinotecan, a high and prolonged intratumoral level of irinotecan, and a greater rate of tumor necrosis at 24 h. Further evaluation of the clinical benefit of DEBIRI is warranted.

Comparative Study of Chemoembolization, Loadable Beads: In vitro Drug Release and Physical Properties of DC Bead and Hepasphere, Loaded with Doxorubicin and Irinotecan

Purpose To characterize in vitro the loadability, physical properties, and release of irinotecan and doxorubicin from two commercially available embolization microspheres. Materials and Methods DC Bead (500-700 μm) and Hepasphere (400-600 μm) microspheres were loaded with either doxorubicin or irinotecan solutions. Drug amount was quantified with spectrophotometry, bead elasticity was measured under compression, and bead size and loading homogeneity were assessed with microscopy image analysis. Drug release was measured over 1-week periods in saline by using a pharmacopeia flow-through method. Results Almost complete drug loading was obtained for both microsphere types and drugs. Doxorubicin-loaded DC Beads maintained their spherical shape throughout the release. In contrast, Hepaspheres showed less homogeneous doxorubicin loading and, after release, some fractured microspheres. Incomplete doxorubicin release was observed in saline over 1 week (27% ± 2 for DC beads and 18% ± 7 for Hepaspheres; P = .013). About 75% of this amount was released within 2.2 hours for both beads. For irinotecan, complete release was obtained for both types of beads, in a sustained manner over 2-3 hours for DC Beads, and in a significantly faster manner as a 7-minute burst for Hepaspheres. Conclusions The two drug-eluting microspheres could be efficiently loaded with both drugs. Incomplete doxorubicin release was attributed to strong drug-bead ionic interactions. Weaker interactions were observed with irinotecan, which led to faster drug release.

DNA Topoisomerase I Gene Copy Number and mRNA Expression Assessed as Predictive Biomarkers for Adjuvant Irinotecan in Stage II/III Colon Cancer.

PURPOSE: Prospective-retrospective assessment of theTOP1gene copy number andTOP1mRNA expression as predictive biomarkers for adjuvant irinotecan in stage II/III colon cancer. EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded tissue microarrays were obtained from an adjuvant colon cancer trial (PETACC3) where patients were randomized to 5-fluorouracil/folinic acid with or without additional irinotecan.TOP1copy number status was analyzed by fluorescencein situhybridization (FISH) using aTOP1/CEN20 dual-probe combination.TOP1mRNA data were available from previous analyses. RESULTS: TOP1FISH and follow-up data were obtained from 534 patients.TOP1gain was identified in 27% using a single-probe enumeration strategy (≥4TOP1signals per cell) and in 31% when defined by aTOP1/CEN20 ratio ≥ 1.5. The effect of additional irinotecan was not dependent onTOP1FISH status.TOP1mRNA data were available from 580 patients with stage III disease. Benefit of irinotecan was restricted to patients characterized byTOP1mRNA expression ≥ third quartile (RFS: HRadjusted, 0.59;P= 0.09; OS: HRadjusted, 0.44;P= 0.03). The treatment byTOP1mRNA interaction was not statistically significant, but in exploratory multivariable fractional polynomial interaction analysis, increasingTOP1mRNA values appeared to be associated with increasing benefit of irinotecan. CONCLUSIONS: In contrast to theTOP1copy number, a trend was demonstrated for a predictive property ofTOP1mRNA expression. On the basis ofTOP1mRNA, it might be possible to identify a subgroup of patients where an irinotecan doublet is a clinically relevant option in the adjuvant setting of colon cancer.Clin Cancer Res; 22(7); 1621-31. ©2015 AACR.

Irinotecan and oxaliplatin: an overview of the novel chemotherapeutic options for the treatment of advanced colorectal cancer

Colorectal cancer is one of the most frequent malignancies in humans and an important cause of cancer death. Metastatic colorectal cancer remains incurable with available systemic therapeutic options. The most active cytotoxic drug against this malignancy, the antimetabolite 5-fluorouracil, was developed more than forty years ago, and as a single agent produces responses in only 10 to 15% of patients which in general last less than one year. Efforts to ameliorate these poor results resulted in the 5-fluorouracil/leucovorin combination, which enhances response rates about two-fold, without, however, significantly improving survival rates. The recent emergence of a handful of new 5-fluorouracil analogues and folate antagonists, as well as the topoisomerase I inhibitor irinotecan, and the third-generation platinum compound oxaliplatin, is likely to alter this gloomy scenario. These agents are at least as effective as 5-fluorouracil in patients with advanced colorectal carcinoma, both untreated and previously treated with 5-fluorouracil-based regimens. This has led to the approval of irinotecan as second-line treatment for 5-fluorouracil-refractory disease, while the use of oxaliplatin has been suggested for patients having a defective 5-fluorouracil catabolism. Recently, FDA approved the combination of irinotecan with 5-fluorouracil and leucovorin for first-line treatment of advanced colon cancer. Based on the synergistic preclinical antitumor effects of some of these agents, their meaningful single-agent activity, distinct mechanisms of cytotoxicity and resistance, and only partially overlapping toxicity profiles, effective combination regimens are now being developed, which are likely to lead to a new, more hopeful era for patients suffering from advanced colorectal carcinoma.

A importância da seqüência de administração do irinotecan e 5-fluorouracil na inibição da proliferação do carcinoma de cólon humano in vitro

O câncer colorretal é um dos tumores humanos mais freqüentes e a terceira causa de morte relacionada ao câncer no mundo. Apesar de importantes progressos terapêuticos, os resultados na doença avançada ainda são muito modestos. Isto deve-se ao fato de que a droga mais utilizada nesta neoplasia, o antimetabólito 5-fluorouracil (5-FU), foi desenvolvido a mais de 40 anos produzindo taxas de respostas de somente 10-15%. Recentemente, o inibidor da topoisomerase I irinotecan (CPT-11) demonstrou, em carcinoma de cólon avançado, respostas comparáveis tanto em pacientes não tratados quanto naqueles que tiveram recaída após terapia com 5-FU. Estes resultados justificam a avaliação da combinação 5-FU/CPT-11 nesta doença. Apesar das respostas dos estudos clínicos serem promissoras, a melhor seqüência de administração destes agentes ainda não foi determinada. Neste estudo, avaliamos a combinação CPT-11/5-FU quanto ao aumento da inibição do crescimento celular quando comparada com os agentes sozinhos nas linhagens celulares de carcinoma de cólon humano SW620, HT-29 e SNU-C4. Para isto, as células foram expostas às drogas sozinhas ou a várias combinações e seqüências de dose baixa e fixa (IC20) de um dos agentes na presença de dose alta e seriada do outro. As células foram avaliadas imediatamente após os tratamentos e/ou cultivadas por mais 2 dias em meio de cultura sem drogas através de coloração com sulforodamina B. As interações entre CPT-11 e 5-FU foram avaliadas por um programa de computador que permite calcular os índices de combinação (CIs) das drogas indicando sinergismo, adição, ou antagonismo (CI < 1, = 1, ou > 1, respectivamente). As respostas celulares foram relacionadas com as atividades das enzimas timidilato sintase, topoisomerase I e carboxil esterase, que foram determinadas através de ensaio que mede os sítios de ligação e atividade catalítica da enzima, ensaio de decatenação do DNA e método espectrofotométrico, respectivamente. Estando a toxicidade no DNA envolvida no mecanismo de ação das duas drogas, também relacionamos as respostas celulares com a introdução de danos ao DNA por método fluorescente. Para melhor entendermos as interações entre as drogas, examinamos os efeitos da exposição à IC20, IC50 e/ou IC80 do CPT-11 ou 5-FU por 2 h ou 24 h em alvos celulares possivelmente relacionados com a citotoxicidade destes agentes. Estes incluem: capacidade de reparo por excisão do 10 DNA, distribuição das células nas fases do ciclo celular, integridade da membrana plasmática e formação de complexos DNA-topoisomerase I. Para isto, utilizamos método de incorporação de [3H-metil]timidina, citometria de fluxo, liberação de lactato desidrogenase (LDH) no meio de cultura e ensaio de precipitação com SDS, respectivamente. Os estudos de inibição do crescimento celular revelaram valores de IC50 do 5-FU nas linhagens SW620, HT-29 e SNU-C4 de aproximadamente 15, 8 e 2 µM, respectivamente, e do CPT-11 próximos a 2, 2 e 4 µM, respectivamente. As diferentes sensibilidades ao 5-FU nas três linhagens foram determinadas principalmente pela diferença na afinidade ao substrato. As respostas comparáveis obtidas pela exposição ao CPT-11 podem ser explicadas pelo equilíbrio entre as diferentes atividades das enzimas topoisomerase I e carboxil esterase, entre as linhagens. O programa de análise da combinação das drogas mostrou adição ou sinergismo após exposição a IC20 do CPT-11 seguido do 5-FU, nas três linhagens celulares. Por outro lado, o pré-tratamento com IC20 do 5-FU antagonizou a inibição do crescimento mediada pelo CPT-11. Nenhum dos tratamentos simultâneos determinou um aumento na inibição do crescimento nas linhagens SW620 e HT-29; mas mostraram adição ou antagonismo na linhagem SNU-C4. Observamos um significativo acréscimo na introdução de danos ao DNA nas linhagens celulares SW620 e HT-29 somente quando a IC20 do CPT-11 precedeu a IC50 do 5-FU. Já na linhagem SNU-C4, não somente este tratamento, mas também a utilização simultânea dos agentes introduziu mais dano ao DNA. Os tratamentos por 2 h ou 24 h com IC20, IC50, e/ou IC80 do 5-FU ou CPT-11 não causaram mudanças significativas na distribuição das células nas fases do ciclo celular, concentração de LDH no meio de cultura, ou formação de complexos DNA-topoisomerse I. Indicando que alterações nestes processos não estão envolvidas com os efeitos modulatórios dos pré-tratamentos na citotoxicidade das drogas. Entretanto, a incorporação de [3H-metil]timidina no DNA de células tratadas com CPT-11, aumentou em função da dose e tempo de exposição à droga. Este resultado sugere que o dano ao DNA introduzido pelo CPT-11 depende da dose e tempo de exposição podendo ser reparado por mecanismos de excisão. Somente foi observada significativa incorporação de [3H-metil]timidina em células tratadas com IC20 do 5-FU por 2 h, sugerindo que o reparo por excisão só ocorre após exposição por curto período e à doses baixas deste agente. Juntos, os resultados deste estudo mostraram que tanto os efeitos anti-proliferativos quanto a introdução de danos ao DNA pela combinação CPT-11/5-FU em dose baixa e fixa de um agente junto com dose alta do outro, nas linhagens celulares de carcinoma de cólon humano SW620, HT-29 e SNU-C4, dependem da seqüência de administração das drogas. Estes achados podem ter sido determinados pelos diferentes efeitos do tratamento com uma dose baixa dos agentes nos mecanismos de reparo por excisão do DNA.

Involvement of nitric oxide on the pathogenesis of irinotecan-induced intestinal mucositis: role of cytokines on inducible nitric oxide synthase activation

Purpose Intestinal mucositis and the closely associated diarrhea are common costly side effects of irinotecan. Cytokine modulators, such as thalidomide and pentoxifylline, are found capable of attenuating intestinal mucositis progression. Nitric oxide (NO) seems to be a key mediator of the antineoplastic drug toxicity. The aim of this study was to investigate the role of NO on the pathogenesis of intestinal mucositis, as well as the participation of cytokines upon inducible nitric oxide synthase (iNOS) expression in irinotecan-induced intestinal mucositis. Methods iNOS-knockout (iNOS(-/-)) and C57BL/6 (WT, wild type) animals (n = 5-6) were given either saline or irinotecan (60 mg/kg i.p for 4 days), with or without pretreatment with aminoguanidine (50 mg/kg s.c.), thalidomide (60 mg/kg s.c), infliximab (5 mg/kg i.v.), or pentoxifylline (1.7 mg/kg s.c). On day 5, diarrhea was assessed, and following euthanasia, proximal intestinal samples were obtained for myeloperoxidase (MPO) and iNOS activity, morphometric analysis, western blot and immunohistochemistry to iNOS, cytokine dosage, and for in vitro evaluation of gut contractility. Results Irinotecan induced severe diarrhea and intestinal smooth muscle over-contractility, accompanied with histopathological changes. Additionally, increased MPO and iNOS activity and iNOS immunoexpression were found in WT animals treated with irinotecan. The rise in MPO, smooth muscle over-contractility, and diarrhea were abrogated in aminoguanidine-treated and iNOS(-/-) mice. Moreover, through western blot, we verified that infliximab and pentoxifylline significantly inhibited irinotecan-induced iNOS expression. In addition, cytokine concentration was found only partially decreased in irinotecan-treated iNOS(-/-) mice when compared with wild-type animals that were given irinotecan. Conclusions This study suggests a role of nitric oxide in the pathogenesis of irinotecan-induced intestinal mucositis and also provides evidence for the participation of cytokines on iNOS induction.

Reversal of established lupus nephritis and prolonged survival of New Zealand black x New Zealand white mice treated with the topoisomerase I inhibitor irinotecan

Systemic lupus erythematosus is a chronic autoimmune disorder that predominantly affects women of childbearing age. Lupus-associated glomerulonephritis is a major cause of mortality in these patients. Current treatment protocols for systemic lupus erythematosus include cyclophosphamide, prednisolone, azathioprine, and mycophenolate mofetil. However, in mice none of these agents alone or in combination were shown to reverse established proteinuria. Using New Zealand Black x New Zealand White F1 mice, we report that administration of the topoisomerase I inhibitor irinotecan from week 13 completely prevented the onset of proteinuria and prolonged survival up to at least 90 wk without detectable side effects. Furthermore, application of irinotecan to mice with established lupus nephritis, as indicated by grade 3+ (> or =300 mg/dl) and grade 4+ (> or =2000 mg/dl) proteinuria and, according to a median age of 35 wk, resulted in remission rates of 75% and 55%, respectively. Survival was significantly prolonged with 73 wk (grade 3+ and 4+ combined) versus 40 wk for control animals. Although total IgG and anti-dsDNA Abs in the serum and mesangial IgG deposits in the kidneys were not reduced in irinotecan-treated mice, subendothelial immune deposits were considerably diminished, suggesting a prevention of glomerular basement membrane disruption. This effect was accompanied by increased rates of ssDNA breaks and inhibition of renal cell apoptosis being different to what is known about irinotecan in anticancer therapy. In conclusion, our data provide evidence that irinotecan might represent an entirely new strategy for the treatment of systemic lupus erythematosus.

Low dose irinotecan improves advanced lupus nephritis in mice potentially by changing DNA relaxation and anti–double-stranded DNA binding

Objective Albeit clear advances in the treatment of SLE, many patients still present with refractory lupus nephritis requiring new treatment strategies for this disease. Here we determined whether reduced doses of the topoisomerase I inhibitor irinotecan, which is known as chemotherapeutic agent, were able to suppress SLE in NZB/W F1 mice. We further evaluated the potential mechanism how irinotecan influenced the course of SLE. Methods NZB/W F1 mice were treated with low dose irinotecan either from week 24 of age or from established glomerulonephritis defined by a proteinuria ≥grade 3+. Binding of anti-dsDNA antibodies was measured by ELISA; and DNA relaxation was visualized by gel electrophoresis. Results Significantly reduced irinotecan dosages improved lupus nephritis and prolonged survival in NZB/W F1 mice. The lowest dose successfully used for the treatment of established murine lupus nephritis was more than 50 times lower than the dose usually applied for chemotherapy in humans. As a mechanism, low dose irinotecan reduced B cell activity; however, the levels of B cell activity in irinotecan-treated mice were similar to those in Balb/c mice of the same age suggesting that irinotecan did not induce a clear immunosuppression. In addition, incubation of double-stranded (ds) DNA with topoisomerase I increased binding of murine and human anti-dsDNA antibodies showing for the first time that relaxed DNA is more susceptible to anti-dsDNA antibody binding. This effect was reversed by addition of the topoisomerase I inhibitor camptothecin. Conclusion Our results propose topoisomerase I inhibitors as a novel and targeted therapy for SLE. © 2014 American College of Rheumatology.

The Topoisomerase I Inhibitor Irinotecan and the Tyrosyl-DNA Phosphodiesterase 1 Inhibitor Furamidine Synergistically Suppress Murine Lupus Nephritis

OBJECTIVE The treatment of lupus nephritis is still an unmet medical need requiring new therapeutic approaches. Our group found recently that irinotecan, an inhibitor of topoisomerase I (topo I), reversed proteinuria and prolonged survival in mice with advanced lupus nephritis. While irinotecan is known to stabilize the complex of topo I and DNA, the enzyme tyrosyl-DNA phosphodiesterase 1 (TDP-1) functions in an opposing manner by releasing topo I from DNA. Therefore, we undertook this study to test whether the TDP-1 inhibitor furamidine has an additional effect on lupus nephritis when used in combination with irinotecan. METHODS NZB/NZW mice were treated with low-dose irinotecan and furamidine either alone or in combination beginning at age 26 weeks. DNA relaxation was visualized using gel electrophoresis. Binding of anti-double-stranded DNA (anti-dsDNA) antibodies to DNA modified by topo I, TDP-1, and the topo I inhibitor camptothecin was determined by enzyme-linked immunosorbent assay. RESULTS Compared to treatment with either agent alone, simultaneous treatment with low-dose irinotecan and furamidine significantly improved survival of NZB/NZW mice. Similar to what has been previously shown for irinotecan alone, the combination treatment did not change the levels of anti-dsDNA antibodies. In vitro, recombinant TDP-1 increased topo I-mediated DNA relaxation, resulting in enhanced binding of anti-dsDNA antibodies. In combination with topo I and camptothecin, TDP-1 reversed the inhibitory effects of camptothecin on DNA relaxation and anti-dsDNA binding. CONCLUSION Affecting DNA relaxation by the enzymes topo I and TDP-1 and their inhibitors may be a promising approach for the development of new targeted therapies for systemic lupus erythematosus.