A semimechanistic-physiologic population pharmacokinetic/pharmacodynamic model for neutropenia following pemetrexed therapy

Purpose: The objectives of these analyses were to (1) develop a semimechanistic-physiologic population pharmacokinetic/pharmacodynamic (PK/PD) model to describe neutropenic response to pemetrexed and to (2) identify influential covariates with respect to pharmacodynamic response. Patients and methods: Data from 279 patients who received 1,136 treatment cycles without folic acid or vitamin B₁₂ supplementation during participation in one of eight phase II cancer trials were available for analysis. Starting doses were 500 or 600 mg pemetrexed per m² body surface area (BSA), administered as 10-min intravenous infusions every 21 days (1 cycle). The primary analyses included 105 patients (279 cycles) for which selected covariates—including vitamin deficiency marker data (i.e., homocysteine, cystathionine, methylmalonic acid, and methylcitrate [I, II, and total] plasma concentrations)—were available. Classical statistical multivariate regression analyses and a semimechanistic-physiologic population PK/PD model were used to evaluate neutropenic response to single-agent pemetrexed administration. Results: The timecourse of neutropenia following single-agent pemetrexed administration was adequately described by a semimechanistic-physiologic model. Population estimates for system-based model parameters (i.e., baseline neutrophil count, mean transit time, and the feedback parameter), which mathematically represent current understanding of the process and physiology of hematopoiesis, were consistent with previously reported values. The population PK/PD model included homocysteine, cystathionine, albumin, total protein, and BSA as covariates relative to neutropenic response. Conclusion: These results support the programmatic decision to introduce folic acid and vitamin B₁₂ supplementation during pemetrexed clinical development as a means of normalizing patient homocysteine levels, thereby managing the risk of severe neutropenia secondary to pemetrexed administration. The current results also suggest that the addition of vitamin B₆ supplementation to normalize patient cystathionine levels may further decrease the incidence of grade 4 neutropenia following pemetrexed administration. The results also suggest the use of folic acid as a means of lessening hematologic toxicity following administration of cytotoxic agents other than antifolates. James J Rusthoven, Robert D Johnson were employees of Eli Lilly and Company, Indianapolis, USA, at the time this work was completed     

Deoxyuridine triphosphatase (dUTPase) expression and sensitivity to the thymidylate synthase (TS) inhibitor ZD9331

Uracil DNA misincorporation and misrepair of DNA have been recognized as important events accompanying thymidylate synthase (TS) inhibition. dUTPase catalyses the hydrolysis of dUTP to dUMP, thereby maintaining low intracellular dUTP. We have addressed the relationship between dUTPase expression and cellular sensitivity to TS inhibition in four human lung tumour cell lines. Sensitivity (5-day MTT assay) to the growth inhibitory effects of the non-polyglutamatable, specific quinazoline TS inhibitor ZD9331, varied up to 20-fold (IC(50)3-70 nM). TS protein expression correlated with TS activity (r(2)= 0.88, P = 0.05). Intracellular concentrations of drug following exposure to ZD9331 (1 microM, 24 h) varied by approximately 2-fold and dTTP pools decreased by > 80% in all cell lines. No clear associations across the cell lines between intracellular drug concentrations, TS activity/expression, or TTP depletion could be made. dUTPase activity varied 17-fold and correlated with dUTPase protein expression (r(2)= 0.94, P = 0.03). There was a striking variation in the amount of dUTP formed following exposure to ZD9331 (between 1.3 and 57 pmole 10(-6)cells) and was in general inversely associated with dUTPase activity. A large expansion in the dUTP pool was associated with increased sensitivity to a 24-h exposure to ZD9331 in A549 cells that have low dUTPase activity/expression. dUTPase expression and activity were elevated (approximately 3-fold) in two variants of a human lymphoblastoid cell line with acquired resistance to TS inhibitors, further suggesting an important role for this enzyme in TS inhibited cells.     

Relationship between homocysteine and neopterin concentrations in patients with gynecological cancer

Elevated concentrations of vascular risk factor homocysteine have been described in patients with malignant diseases, and homocysteine was supposed to be useful as tumor marker. Likewise, elevated concentrations of Th1-type immune activation marker neopterin are frequently observed in patients suffering from cancer and serve as prognostic marker for the survival of patients. In this study, the relationship between homocysteine and neopterin concentrations was examined in 18 patients with gynecological cancer. Concentrations of homocysteine and cysteine were measured by HPLC in sera of patients, folic acid and vitamin B(12) levels were determined by radioimmunoassay, and neopterin concentrations were measured by ELISA. Median homocysteine concentration was 11.2 microM (interquartile range: 9.9-13.2 microM), 3 patients had levels higher than 15 microM, the upper limit of the normal range. Neopterin concentrations were increased in 13 patients (median: 11.6; 7.7-24.9 nM), cysteine (median: 234; 216-255 microM), folate (median: 7.8; 6.2-11.7 ng/ml) and vitamin B(12) (median: 352; 258-570 pg/ml) concentrations were all within reference ranges although rather at the lower side. Higher homocysteine concentrations correlated inversely with low folate concentrations (r(s)=-0.605; P<0.01) and tended to be higher in patients with higher cysteine levels (r(s)=0.457; P<0.06; ). No correlation was found between homocysteine and immune activation marker neopterin, although the three patients with elevated homocysteine concentrations tended to have higher neopterin levels as well (P<0.07). In conclusion, only a few patients with gynecological cancer present with elevated homocysteine concentrations and hyperhomocysteinemia seems only weakly related to immune activation phenomena, tumor cell proliferation probably is more important for the increase of homocysteine.     

Dynamics of antifolate transport via the reduced folate carrier and the membrane folate receptor in murine leukaemia cells in vitro and in vivo

Murine L1210 leukaemia cells expressing either the reduced folate carrier (RFC) or the membrane folate receptor (MFR) were studied in vitro and in vivo to assess the dynamics of membrane transport of two categories antifolates; folate-based inhibitors of dihydrofolate reductase (methotrexate, edatrexate, aminopterin, PT523, and PT644) and thymidylate synthase (TS) [CB3717, raltitrexed, plevitrexed (BGC9331), pemetrexed and GW1843]. The potency of in situ inhibition of TS was used as an endpoint to analyze the in vitro dynamics of RFC/MFR-membrane transport of these antifolates. Both for L1210-RFC and L1210-MFR cells, the potency of in situ TS inhibition was closely correlated with increasing affinities of these transporters for the antifolates (r = 0.64, P < 0.05 and r = −0.65, P < 0.05, respectively). Within the group of antifolates for which MFR had a low binding affinity, those that had the ability to become polyglutamylated, were more potent inhibitors of TS in situ activity than non-polyglutamatable antifolates. In vivo activity of methotrexate, edatrexate, raltitrexed and pemetrexed was assessed in L1210-RFC and L1210-MFR bearing mice that were fed either a standard or a folate-deficient chow. Dietary folate depletion significantly reduced the MTD for methotrexate (sevenfold), edatrexate (sevenfold), raltitrexed (50-fold) and pemetrexed (150-fold). Based on increased life spans, antitumor effects of methotrexate and edatrexate were markedly better in L1210-RFC bearing mice on the folate-deficient chow (ILS: 455 and 544%, respectively) than on standard chow (ILS: 213 and 263%, respectively). No therapeutic effects of methotrexate and edatrexate were observed for L1210-MFR bearing mice on either chow condition, which may be consistent with the low binding affinity for MFR. Irrespective of the folate diet status, pemetrexed and raltitrexed were inactive against both L1210-RFC and L1210-MFR bearing mice, which may be due to high circulating plasma thymidine levels. Collectively, this study underscores that modulation of dietary folate status can provide a basis within which the therapeutic effect of antifolates may be further improved.     

TS和RRM1表达与培美曲塞、吉西他滨联合卡铂治疗晚期非鳞非小细胞肺癌疗效的关系

目的 探讨胸苷酸合成酶(TS)和核苷酸还原酶M1(RRM1)的表达与培美曲塞、吉西他滨联合卡铂治疗晚期非鳞非小细胞肺癌疗效的关系.方法 选取晚期非鳞非小细胞肺癌患者98例,其中接受培美曲塞联合卡铂治疗患者49例(A组),接受吉西他滨联合卡铂治疗患者49例(B组).免疫组化检测所有肿瘤标本中TS和RRM1的表达,并分析其表达与化疗疗效的关系.结果 培美曲塞组TS-患者总有效率为64.3％,显著高于同组TS+患者总有效率42.9％,差异具统计学意义(P＜0.05);培美曲塞组RRM1+和RRM1-患者总有效率分别为52.1％和57.7％,差异无统计学意义(P＞0.05).吉西他滨组RRM1-患者总有效率为57.7％,显著高于同组RRM1+患者总有效率47.8％,差异具统计学意义(P＜0.05);吉西他滨组TS+和TS-患者总有效率分别为50.0％和56.0％,差异无统计学意义(P＞0.05).培美曲塞组TS-患者胃肠道毒副反应和血液毒副反应发生率分别为17.9％和21.4％,显著低于TS+患者(P＜0.05);吉西他滨RRM1-患者胃肠道毒副反应和血液毒副反应发生率分别为23.1％和26.9％,显著低于RRM1+患者(P＜0.05).2组患者不良反应总发生率相比较,培美曲塞组患者相对能较好耐受,差异具统计学意义(P＜0.05).培美曲塞组患者FACT-L评分显著优于吉西他滨组患者,差异具统计学意义(P＜0.05).结论 TS-患者接受培美曲塞治疗有较好临床获益,RRM1-患者接受吉西他滨治疗临床荻益较好,且培美曲塞化疗方案相对吉西他滨有较好临床耐受性,毒副作用相对较低,治疗后患者FACT-L评分较高.     

Significance of thymidylate synthase for resistance to pemetrexed in lung cancer

Pemetrexed (MTA) is a multitargeted antifolate with promising clinical activity in lung cancer. We exposed the small cell lung cancer cell line PC6 to stepwise-increasing pemetrexed concentrations of 0.4, 1.6, and 4.0 μ m , and established three pemetrexed-resistant lung cancer cell lines: PC6/MTA-0.4, PC6/MTA-1.6, and PC6/MTA-4.0 cells. To investigate the mechanisms of acquired resistance to pemetrexed, we measured the expression levels of the thymidylate synthase ( TS ), reduced folate carrier ( RFC ), and folylpoly-gamma-glutamate synthetase ( FPGS ) genes. TS gene expression was significantly increased in PC6/MTA-1.6 and PC6/MTA-4.0 cells relative to parental cells in a pemetrexed dose-dependent manner. In contrast, the levels of RFC gene expression in PC6/MTA-0.4 cells and FPGS in PC6/MTA-1.6 cells were significantly decreased, whereas the levels of both genes were restored in PC6/MTA-4.0 cells. Knockdown of TS expression using siRNA enhanced pemetrexed cytotoxicity in PC6/MTA-4.0 cells. The expression level of the TS gene was significantly correlated with the concentration of pemetrexed for 50% cell survival (IC₅₀) in 11 non-small cell lung cancer cell lines. These results suggest that the alteration of molecular pharmacological factors in relation with pemetrexed resistance is dose-dependent, and that up-regulation of the expression of the TS gene may have an important role in the acquired resistance to pemetrexed. In addition, TS may be a predictive marker for pemetrexed sensitivity in lung cancer. ( Cancer Sci 2009)     

Randomized phase 2 trial on refinement of early-stage NSCLC adjuvant chemotherapy with cisplatin and pemetrexed versus cisplatin and vinorelbine: the TREAT study

BackgroundAdjuvant chemotherapy is beneficial in non-small-cell lung cancer (NSCLC). However, balancing toxicity and efficacy mandates improvement.Patients and methodsPatients with completely resected stages IB-pT3N1 NSCLC were randomly assigned to either four cycles cisplatin (C: 50 mg/m² day (d)1 + 8) and vinorelbine (V: 25 mg/m² d1, 8, 15, 22) q4 weeks or four cycles cisplatin (75 mg/m² d1) and pemetrexed (Px: 500 mg/m² d1) q3 weeks. Primary objective was the clinical feasibility rate (no grade (G)4 neutropenia/thrombocytopenia or thrombocytopenia with bleeding, no G3/4 febrile neutropenia or non-hematological toxicity; no premature withdrawal/death). Secondary objectives were drug delivery and efficacy.ResultsOne hundred and thirty two patients were randomized (stages: 38% IB, 10% IIA, 47% IIB, 5% pT3pN1; histology: 43% squamous, 57% non-squamous). The feasibility rates were 95.5% (cisplatin and pemetrexed, CPx) and 75.4% (cisplatin and vinorelbine, CVb) (P = 0.001); hematological G3/4 toxic effects were 10% (CPx) and 74% (CVb) (P < 0.001), non-hematological toxic effects were comparable (33% and 31%, P = 0.798). Delivery of total mean doses was 90% of planned with CPx, but 66% (cisplatin) and 64% (vinorelbine) with CVb (P < 0.0001). The median number of cycles [treatment time (weeks)] was 4 for CPx (11.2) and 3 for CVb (9.9). Time to withdrawal from therapy differed significantly between arms favoring CPx (P < 0.001).ConclusionAdjuvant chemotherapy with CPx is safe and feasible with less toxicity and superior dose delivery compared with CVb.     

Suicide prodrugs activated by thymidylate synthase: rationale for treatment and noninvasive imaging of tumors with deoxyuridine analogues.

Most tumors are resistant to therapy by thymidylate synthase (TS) inhibitors due to their high levels of TS. Instead of inhibiting TS, we hypothesized that it was possible to use this enzyme to activate suicide prodrugs (deoxyuridine analogues) to more toxic species (thymidine analogues). Tumors with high levels of TS could be particularly sensitive to deoxyuridine analogues because they would be more efficient in producing the toxic methylated species. Furthermore, the accumulation of methylated species within tumors could be visualized externally if a tracer dose of the deoxyuridine analogue was tagged with an isotope, preferably a positron emitter, such as 18F. Higher accumulation of isotope indicates higher activity of TS and lower sensitivity of the tumor to TS inhibitors, but perhaps more sensitivity to therapy with deoxyuridine analogues as suicide prodrugs. 2'-F-ara-deoxyuridine (FAU) was used as a prototype to demonstrate these concepts experimentally. FAU readily entered cells and was phosphorylated, methylated, and subsequently incorporated into cellular DNA. Among different cell lines, FAU produced varying degrees of growth inhibition. Greater DNA incorporation (e.g., for CEM and U-937 cells) was reflected as increased toxicity. FAU produced less DNA incorporation in Raji or L1210 cells, and growth rate was minimally decreased. As the first demonstration that cells with high levels of TS activity can be more vulnerable to therapy than cells with low TS activity, this preliminary work suggests a new therapeutic approach for common human tumors that were previously resistant. Furthermore, it appears that the TS activity of tumors could be noninvasively imaged in situ by tracer doses of [18F]FAU and that this phenotypic information could guide patient therapy.     

Rapamycin downregulates thymidylate synthase and potentiates the activity of pemetrexed in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) accounts for 80–85% of lung cancer cases, and almost half of newly diagnosed patients have metastatic disease. Pemetrexed is a widely used drug for NSCLC and inhibits several folate-dependent enzymes including thymidylate synthase (TS). Increased expression of TS confers resistance to pemetrexed in vitro and predicts poor response to pemetrexed. Rapamycin is an mTOR inhibitor and suppresses cap-dependent synthesis of specific mRNA species. Here, we show that the combination of rapamycin and pemetrexed synergistically inhibits proliferation of NSCLC cells. Although pemetrexed as a single agent induced TS, pretreatment with rapamycin suppressed pemetrexed-induced TS expression. In vivo, the combination of rapamycin and pemetrexed inhibited growth of NSCLC xenografts, which correlated with decreased mTOR activity and suppression of pemetrexed-induced TS expression. The ability of rapamycin to enhance the efficacy of pemetrexed and prevent TS expression has implications for the design of Phase I and/or Phase II NSCLC clinical trials with mTOR inhibitors in combination with pemetrexed.     

Thymidylate synthase and dihydrofolate reductase expression in non-small cell lung carcinoma: the association with treatment efficacy of pemetrexed

Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are target enzymes of inhibition by pemetrexed, an antifolate for treatment of advanced non-small-cell lung cancer (NSCLC). This study is to evaluate the association of TS and DHFR expressions and the treatment efficacy of pemetrexed in NSCLC patients. From January 2006 to October 2008, patients with advanced NSCLC treated with pemetrexed after prior chemotherapy were included. The TS and DHFR expressions in tumor tissues were examined by immunohistochemistry and evaluated by a semiquantitative histologic score (H-score). The H-score was derived from the degrees of intensity of tumor cells multiplied by the percentage of positive neoplastic cells. The medical records were reviewed and analyzed with respect to patients’ characteristics, histology types, treatment responses and survivals. Among 268 NSCLC patients treated with pemetrexed, 49 had tumor specimens available for TS and DHFR evaluation. The TS expression was positively correlated with DHFR expression (r² = 0.11, p = 0.02). Patients with low TS (≤150) expression had a longer median progression-free survival (PFS) than those with high TS (>150) expression (4.8 vs. 3.4 months; p = 0.01). Patients with low DHFR expression (≤120) also had a longer median PFS than patients with high DHFR expression (>120), which was not statistically significant (5.8 vs. 3.6 months; p = 0.33). In patients with adenocarcinoma, the low TS patient group also had a longer median PFS and a longer median overall survival (OS) as compared with patients with high TS expression (PFS, 4.8 vs. 3.8 months, p = 0.03; OS, 21.4 vs. 10.0 months, p = 0.03). Nevertheless, the association of DHFR expression level and median PFS as well as OS were not statistically significant. TS expression, rather than DHFR, may be an important predictive factor for treatment efficacy of pemetrexed in NSCLC patients.     

Thymidylate synthase as a determinant of pemetrexed sensitivity in non-small cell lung cancer

Background:

Although a high level of thymidylate synthase (TS) expression in malignant tumours has been suggested to be related to a reduced sensitivity to the antifolate drug pemetrexed, no direct evidence for such an association has been demonstrated in non-small cell lung cancer (NSCLC). We have now investigated the effect of TS overexpression on pemetrexed sensitivity in NSCLC cells.

Methods:

We established NSCLC cell lines that stably overexpress TS and examined the effects of such overexpression on the cytotoxicity of pemetrexed both in vitro and in xenograft models. We further examined the relation between TS expression in tumour specimens from NSCLC patients and the tumour response to pemetrexed by immunohistochemical analysis.

Results:

The sensitivity of NSCLC cells overexpressing TS to the antiproliferative effect of pemetrexed was markedly reduced compared with that of control cells. The inhibition of DNA synthesis and induction of apoptosis by pemetrexed were also greatly attenuated by forced expression of TS. Furthermore, tumours formed by TS-overexpressing NSCLC cells in nude mice were resistant to the growth-inhibitory effect of pemetrexed observed with control tumours. Finally, the level of TS expression in tumours of non-responding patients was significantly higher than that in those of responders, suggestive of an inverse correlation between TS expression and tumour response to pemetrexed.

Conclusion:

A high level of TS expression confers a reduced sensitivity to pemetrexed. TS expression is thus a potential predictive marker for response to pemetrexed-based chemotherapy in NSCLC patients.     

培美曲塞联合顺铂与长春瑞滨联合顺铂一线治疗晚期NSCLC的对比研究

目的 比较培美曲塞或长春瑞滨联合顺铂一线治疗晚期非小细胞肺癌（NSCLC）的疗效及毒副反应。方法 回顾性分析我院2008年1月至2010年12月收治的68例晚期NSCLC患者,分别接受培美曲塞联合顺铂（PC方案组,32例）或长春瑞滨联合顺铂（NP方案组,36例）一线治疗。PC方案组：培美曲塞500mg／m2,d1;顺铂 25mg／m²,d₁～d₃。NP方案组：长春瑞滨 25mg／m²,d₁、d₈,顺铂25mg／m²,d₁～d₃。每3周为1周期,每2个周期评价疗效。结果 所有患者均可评价近期疗效。两组均无完全缓解病例,PC方案组与NP方案组的有效率（RR）分别为40.6%（13/32）和36.1%（13/36）,疾病控制率（DCR）分别为71.9%（23/32）和61.1%（22/36）,两组RR和DCR的差异均无统计学意义（P＞0.05）。两组中位疾病进展时间（TTP）分别为6.2和5.2个月,组间差异无统计意义（P＞0.05）。NP方案组3～4级白细胞减少、中性粒细胞减少的发生率高于PC方案组（P＜0.05）。 结论 培美曲塞联合顺铂与长春瑞滨联合顺铂一线治疗晚期NSCLC的疗效相当,但培美曲塞的毒副反应较少。     

Cellular and molecular mechanisms for the synergistic cytotoxicity elicited by oxaliplatin and pemetrexed in colon cancer cell lines

Purpose

Oxaliplatin effect in the treatment of colorectal cancer is improved upon combination with thymidylate synthase (TS) inhibitors. Pemetrexed is polyglutamated by the folylpolyglutamate synthase (FPGS) and blocks folate metabolism and DNA synthesis by inhibiting TS, dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study evaluates the pharmacological interaction between oxaliplatin and pemetrexed in colorectal cancer cells.

Methods

Human HT29, WiDr, SW620 and LS174T cells were treated with oxaliplatin and pemetrexed. Drug interaction was studied using the combination index method, while cell cycle was investigated with flow cytometry. The effects of drugs on Akt phosphorylation and apoptosis were studied with ELISA and fluorescence microscopy, respectively. RT-PCR analysis was performed to assess whether drugs modulated the expression of pemetrexed targets and of genes involved in DNA repair (ERCC1 and ERCC2). Finally, platinum–DNA adduct levels were detected by ultra-sensitive multi-collector inductively coupled plasma mass spectrometry (ICP-MS).

Results

A dose-dependent inhibition of cell growth was observed after drug exposure, while a synergistic interaction was detected preferentially with sequential combinations. Oxaliplatin enhanced cellular population in the S-phase. Drug combinations increased apoptotic indices with respect to single agents, and both drugs inhibited Akt phosphorylation. RT-PCR analysis showed a correlation between the FPGS/(TS × DHFR × GARFT) ratio and pemetrexed sensitivity, as well as a downregulation of ERCC1, ERCC2, TS, DHFR and GARFT after drug exposure. In addition, pretreatment with pemetrexed resulted in an increase of oxaliplatin–DNA adducts.

Conclusion

These data demonstrate that oxaliplatin and pemetrexed synergistically interact against colon cancer cells, through modulation of cell cycle, inhibition of Akt phosphorylation, induction of apoptosis and modulation of gene expression.     

Biochemical effects of folate-based inhibitors of thymidylate synthase in MGH-U1 cells

The TS-inhibitory effects induced by a 24-h exposure to the folate-based TS inhibitors CB3717, C2-desamino analogs of CB3717 including D1694, and BW1843U89 were quantitated using the MGH-U1 human bladder carcinoma. The effects of D1694 on the time course of TS inhibition and on intracellular deoxyuridine monophosphate (dUMP) accumulation and deoxyuridine (dUrd) production were evaluated. D1694 and BW1843U89 were the most active TS inhibitors with IC₅₀ values of 2.4 and 0.5 nM, respectively. The C2-desamino C2-methyl dideazafolates were 27–292 times more potent than the parent CB3717 as TS inhibitors. A methyl group at the C2 position of CB3717 had the most dramatic effect, whereas a thiazole substitution for a benzyl added a small benefit and N10 substitution had a limited impact on TS-inhibitory potency and clonogenic survival. There was a significant correlation between the IC₅₀ values for TS inhibition and those for cytotoxic potency obtained for these drugs. LV and thymidine protected cells from these folate-based TS inhibitors. Intracellular dUMP levels following 24 h D1694 (IC₅₀) exposure increased 7-fold. Levels of dUrd effluxing into the media increased up to 4.5 M following a 24-h exposure to D1694 (IC₉₀). We conclude that (a) C2-desamino C2-methyl dideazafolates are potent TS inhibitors, (b) TS inhibition requires prolonged exposure with these folate TS inhibitors, (c) survival is correlated with inhibition of TS for the folate-based TS inhibitors and (d) the biochemical consequences of TS inhibition include increased dUMP and dUrd levels.Abbreviations TS thymidylate synthase - D1694 N-{5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl}-L-glutamic acid - BW1843U89 (S)-2-(5-[{1,2-dihydro-3-methyl-1-oxo-benzo(F)quinazolin-9-yl]-methyl}amino]1-oxo-2-isoindolinyl) glutamic acid - CB3717 N ¹⁰-propargyl-5,8-dideazafolic acid - dCB3717 2-desamino-N ¹⁰-propargyl-5,8-dideazafolic acid - LV leucovorin - FPGS folylpolyglutamate synthetase - IC ₅₀ IC ₉₀ concentrations necessary to inhibit function by 50% and 90%, respectively This study was supported by a grant from the National Cancer Institute of Canada     

Homocysteine, pharmacogenetics, and neurotoxicity in children with leukemia.

PURPOSE: Despite its clinical success, methotrexate (MTX) therapy is associated with toxicities such as seizures, the pathogenesis of which remains unclear. It has been suggested that hyperhomocysteinemia is caused by MTX and is responsible for its neurotoxic effects. The purposes of this study were to explore whether hyperhomocysteinemia was related to MTX administration and toxicity and whether homocysteine or MTX toxicity differed by methylenetetrahydrofolate reductase (MTHFR) or reduced folate carrier (RFC) genetic polymorphisms. PATIENTS AND METHODS: We studied 53 children with newly diagnosed acute lymphoblastic leukemia who were consecutively treated on a single clinical protocol that included two courses of high-dose MTX (high-dose methotrexate [HDMTX]; 2.5 or 5.0 g/m2 per day) as consolidation therapy. RESULTS: The study participants' median plasma homocysteine concentrations at 23 and 44 hours after HDMTX (9.00 micromol/L and 10.12 micromol/L, respectively) were greater than the concentrations immediately before HDMTX (5.77 micromol/L, P <.0001 for both comparisons). Seven days after HDMTX treatment, their plasma concentration returned to baseline. Nine patients experienced seizures, and five patients experienced thrombosis during the first 15 months of therapy, with a tendency for there to be higher plasma homocysteine in patients with seizures across all time points (P =.063) but not in patients with thrombosis (P =.59). We observed no significant differences in plasma or cerebrospinal fluid homocysteine levels or in toxicity based on the MTHFR 677C/T or RFC 80G/A genotypes. CONCLUSION: We conclude that homocysteine was transiently elevated after HDMTX and may be related to seizure risk in children with leukemia.     

A Prospective Study of Shortened Vitamin Supplementation Prior to Cisplatin–Pemetrexed Therapy for Non‐Small Cell Lung Cancer

Background.

Prior supplementation with folic acid and vitamin B₁₂ is required to reduce pemetrexed therapy toxicity; the recommended lead-in time is at least 7 days. On the basis of previous pharmacokinetic and clinical studies, we hypothesized that the lead-in time could be shortened to 24 hours, enabling earlier commencement of standard chemotherapy; thus, we planned the first prospective trial of this regimen.

Methods.

Patients with advanced nonsquamous non-small cell lung cancer who had not previously received cytotoxic chemotherapy were enrolled. After measurement of homocysteine concentrations, the patients received 1,000 μg of vitamin B₁₂ by intramuscular injection and began taking 350–500 μg of oral folic acid daily. Starting 24–48 hours after the vitamin B₁₂ injection, the patients received intravenous 500 mg/m² pemetrexed and 75 mg/m² cisplatin for 4 cycles at 3 weekly intervals. The primary endpoint was the proportion of patients who developed neutropenia grade ≥3.

Results.

Thirty patients received chemotherapy starting within 48 hours of the vitamin B₁₂ injection. No treatment-related deaths or grade 4 toxicity occurred. Neutropenia grade ≥3, other laboratory toxicities grade ≥3, and nonlaboratory toxicities grade ≥3 occurred in 6.7%, 13%, and 13% of patients, respectively. The baseline homocysteine concentrations were not higher in patients with grade ≥3 toxicities than in the remainder of the cohort (mean values, 8.6 and 10.7 μmol/L, respectively). The response rate to chemotherapy was 43%.

Conclusion.

The shortened vitamin supplementation was well tolerated and retained antitumor efficacy. Analysis of baseline homocysteine concentrations confirmed the efficacy of short-term vitamin supplementation.     

肝癌患者血浆、组织中凝血及纤溶因子的表达及其临床意义

背景与目的:研究表明凝血及纤溶因子改变与恶性肿瘤的发生、发展密切相关。本研究检测组织因子(tissuefactor,TF)、尿激酶型纤溶酶原激活物(urokinase-typeplasminogenactivator,uPA)及其受体(urokinase-typeplasminogenactivatorreceptor,uPAR)在肝细胞癌(hepatocellularcarcinoma,HCC)组织中及血浆中的表达并探讨其临床意义。方法:应用酶联免疫吸附法(ELISA)检测肝细胞癌患者50例及对照组30例非癌患者的血浆TF、uPA和uPAR水平;50例肝癌中随机选取27例取其肝癌组织、癌旁组织,以27例非癌患者肝组织为对照,利用RT-PCR法分别检测TF、uPA、uPARmRNA的阳性率及相对表达强度,并结合临床病理资料进行分析。结果:肝细胞癌患者血浆TF、uPA及uPAR水平均较对照组升高[(409.4±13.0)pg/mlvs.(318.8±69.1)pg/ml,(1.63±0.52)ng/mlvs.(1.20±0.40)ng/ml,(1.36±1.00)ng/mlvs.(0.68±0.28)ng/ml],均有显著性差异(P<0.05)。肝细胞癌患者血浆TF水平在低分化组、肿瘤较大组及合并肝硬化组均显著升高(P<0.05),血浆uPA水平只在合并肝硬化组升高(P<0.05)。肝细胞癌患者血浆TF、uPA和uPAR水平在有淋巴结转移、肝外脏器转移及门脉癌栓组较无转移及无癌栓组升高(P<0.05)。TF、uPA、uPARmRNA在肝细胞癌组织中阳性率及相对表达强度分别为62.96%(17/27)、70.37%(19/27)、77.78%(21/27)及0.57±0.27、0.96±0.46、0.78±0.32,均显著高于癌旁组织及非癌患者肝组织,均有显著性差异(P<0.05);TF、uPA、uPARmRNA在有肝内转移及门脉癌栓组的阳性率及相对表达强度均高于无肝内转移及门脉癌栓组(P<0.05)。经Pearson检验,肝细胞癌患者TF、uPA和uPARmRNA表达呈正相关(TF/uPA:r=0.37,P<0.01,TF/uPAR:r=0.53,P<0.01,uPA/uPAR:r=0.36,P<0.01)。经Cox多因素分析三者均为独立预后因素[TF(χ2=6.05,P=0.014),uPA(χ2=4.29,P=0.038),uPAR(χ2=4.40,P=0.036)]。结论:TF、uPA及uPAR可能在肝细胞癌的侵袭转移过程中起协同作用;三者可能与肝癌患者预后有关。     

培美曲塞和吉非替尼不同时序应用对人肺腺癌细胞的作用和机制

目的 探讨培美曲塞与吉非替尼不同时序应用对肺腺癌细胞A549和PC-9生长及凋亡的影响, 并阐述其可能机制。方法 MTT法检测各组细胞的增殖抑制情况,流式细胞仪检测各组细胞凋亡及细胞周期分布,Western印迹法检测对EGFR下游信号通路及TS酶蛋白水平表达的影响。结果 培美曲塞序贯吉非替尼、培美曲塞同步联合吉非替尼对PC-9和A549细胞增殖抑制率及凋亡率较单药组均提高（P<0.05）,培美曲塞可以提高EGFR、AKT 、ERK磷酸化水平,而吉非替尼表现为抑制作用, 同时吉非替尼降低TS酶表达。培美曲塞序贯吉非替尼,培美曲塞同步联合吉非替尼抑制EGFR、AKT 、ERK磷酸化水平较单药更强。吉非替尼主要将PC-9、A549细胞阻滞在G0/G1期;培美曲塞主要将细胞阻滞在S期。培美曲塞序贯吉非替尼、培美曲塞同步联合吉非替尼较其他组G2/M期细胞比例提高（P<0.05）。结论 培美曲赛序贯吉非替尼、培美曲赛同步联合吉非替尼在PC-9、A549细胞中均起到协同增效作用,且培美曲赛序贯吉非替尼协同作用更为显著,可能主要与培美曲赛诱导EGFR、AKT 、ERK磷酸化及吉非替尼降低TS酶作用有关。     

The longitudinal relationship between circulating concentrations of C-reactive protein, interleukin-6 and interleukin-10 in patients undergoing resection for renal cancer

The systemic inflammatory response, as evidenced by elevated circulating concentrations of C-reactive protein, is a stage-independent prognostic factor in patients undergoing curative nephrectomy for localised renal cancer. However, it is not clear whether the systemic inflammatory response arises from the tumour per se or as a result of an impaired immune cytokine response. The aim of the present study was to examine C-reactive protein, interleukin-6 and interleukin-10 concentrations before and following curative resection of renal cancer. Sixty-four patients with malignant renal disease and 12 with benign disease, undergoing resection were studied. Preoperatively, a blood sample was collected for routine laboratory analysis with a further sample stored before analysis of interleukin-6 and interleukin-10 using an enzyme-linked immunosorbent assay (ELISA) technique. The blood sampling procedure and analyses were repeated at approximately 3 months following resection. Circulating concentrations of both interleukin-6 and interleukin (P< or =0.01) were higher and a greater proportion were elevated (P<0.05) in malignant compared with benign disease. The renal cancer patients were grouped according to whether they had evidence of a systemic inflammatory response. In the inflammatory group T stage was higher (P<0.01), both interleukin-6 and interleukin-10 concentrations were higher (P<0.001) and elevated (P<0.10) compared with the non-inflammatory group. Tumour volume was weakly correlated with C-reactive protein (r(2)=0.20, P=0.002), interleukin-6 (r(2)=0.20, P=0.002) and interleukin-10 (r(2)=0.24, P=0.001). Following nephrectomy the proportion of patients with elevated C-reactive protein, interleukin-6 and interleukin-10 concentrations did not alter significantly. An elevated preoperative C-reactive protein was associated with increased tumour stage, interleukin-6 and interleukin-10 concentrations. However, resection of the primary tumour did not appear to be associated with significant normalisation of circulating concentrations of C-reactive protein, interleukin-6 or interleukin-10. Therefore, the presence of systemic inflammatory response is unlikely to be solely be determined by the tumour itself, but may be as a result of an impaired immune cytokine response in patients with renal cancer.