A phase Ib dose-escalation study of erlotinib, capecitabine and oxaliplatin in metastatic colorectal cancer patients.

BACKGROUND: Dysregulation of the epidermal growth factor receptor (HER1/EGFR) has been reported in colorectal cancer (CRC). Erlotinib is a potent inhibitor of HER1/EGFR-mediated signaling. This trial of patients with metastatic CRC (MCRC) examined the safety, maximum tolerated dose (MTD) and pharmacokinetics (PK) of erlotinib in combination with capecitabine and oxaliplatin (XELOX), a regimen with established efficacy. PATIENTS AND METHODS: Patients previously untreated or treated with one line of 5-fluorouracil and/or irinotecan received escalating oral doses of erlotinib (daily), capecitabine (days 1-14) and i.v. oxaliplatin (day 1 of a 21-day cycle). RESULTS: The first six patients in cohort 1 (erlotinib 100 mg/day, capecitabine 825 mg/m(2) twice daily, oxaliplatin 130 mg/m(2)) had no dose-limiting toxicities (DLTs). In cohort 2 (capecitabine increased to 1000 mg/m(2) twice daily), two of six patients had DLTs. When cohort 2 was expanded to 11 patients two further DLTs occurred, exceeding the definition of MTD. Cohort 1 was expanded to 12 patients, and no DLTs occurred. The most common adverse events (AEs) were diarrhea and rash. There was a trend for reduced capecitabine concentrations in the presence of erlotinib. While this was not statistically significant, the possibility of an interaction affecting capecitabine PK cannot be excluded. Antitumor activity was observed in both cohorts (one complete and four partial responses, and stable disease in 11 patients). CONCLUSION: The MTD for this combination in MCRC is capecitabine 825 mg/m(2) twice daily days 1-14, oxaliplatin 130 mg/m(2) day 1 and erlotinib 100 mg/day of a 21-day cycle. The combination was well tolerated at the MTD, with no unexpected AEs. The use of this combination in MCRC warrants further investigation.

Clinafloxacin monotherapy (CI-960) versus ceftazidime plus amikacin for empirical treatment of febrile neutropenic cancer patients.

OBJECTIVE: To assess the efficacy and safety of clinafloxacin as a single agent for the empirical treatment of febrile episodes and bacterial infections in neutropenic cancer patients. METHODS: An open label, active-controlled, randomized, parallel treatment, multicenter study was conducted where clinafloxacin monotherapy was compared to the combination of ceftazidime plus amikacin (plus optional vancomycin or teicoplanin). Four hundred and nineteen patients were randomized to receive either intravenous clinafloxacin 200 mg every 12 h or intravenous ceftazidime (2 g) iv every 8 h plus intravenous amikacin (15 mg/kg) per day in divided doses. All randomized patients were to receive a minimum of 48 h of primary study drug treatment, after which the primary treatment could be modified. Clinical and microbiological responses were evaluated at 7-21 days post-treatment after study treatment and long term (maximum 28 days), in intent-to-treat and modified intent-to-treat populations. RESULTS: Clinafloxacin and ceftazidime-amikacin were statistically equivalent for the 72-h defervescence rate, overall defervescence rate, time to defervescence, clinical success rate, by-pathogen microbiological eradication rate, and survival rate. Clinical cure was achieved in 84% (59/70) of patients who received clinafloxacin monotherapy. There were no significant differences between treatments in rates of adverse events or treatment discontinuation rates due to adverse events. CONCLUSIONS: Clinafloxacin appears to be an appropriate agent for empirical treatment in febrile neutropenic cancer patients.

Pharmacokinetics of tilidine in terminal renal failure.

The aim of the present study was to investigate the pharmacokinetics of tilidine and its metabolites during the dialysis procedure and in the dialysis-free interval. Tilidine is a prodrug that is metabolized presystemically into the active metabolite nortilidine. Nortilidine is degraded thereafter to bisnortilidine and several polar metabolites. Nine patients with a creatinine clearance < 5 ml/min were treated in a crossover design with single oral doses of 1.5 mg/kg on the day of dialysis (dialysis performed from 3 to 6 hours after drug administration) and on a day in the dialysis-free interval. Blood samples were taken frequently and analyzed for tilidine, nortilidine, and bisnortilidine. Drug and metabolite concentrations were also measured in aliquots of dialysate collected during dialysis. Only negligible amounts of tilidine, nortilidine, and bisnortilidine (about 0.9% of the dose) were recovered from the dialysate. The pharmacokinetics of nortilidine and its inactive metabolite bisnortilidine was not affected by dialysis. The presystemic apparent clearance of the prodrug tilidine was decreased significantly during the dialysis-free interval. A significant decrease of the rate of elimination and an increase of the AUC of bisnortilidine were observed if these parameters were compared with data obtained from healthy volunteers. The plasma concentrations of nortilidine were comparable in patients and normal volunteers. Thus, a reduction of the dose of tilidine in patients with severely impaired kidney function seems not to be required. Tilidine and its metabolites cannot be removed from the body by dialysis.

Pharmacokinetics of nortilidine and naloxone after administration of tilidine/naloxone solution or tilidine/naloxone sustained release tablets.

Valoron N is a compound which consists of the prodrug tilidine (CAS 20380-58-9), from which the active metabolite nortilidine is formed by demethylation in the liver, and the opiate antagonist naloxone (CAS 465-65-6), which prevents the abuse of the analgesic by opiate dependents. The pharmacokinetics of nortilidine and naloxone were studied in 18 male healthy subjects after oral application of tilidine/naloxone solution or tilidine/naloxone retard tablets, respectively. The following report gives the results on investigations of a) dose linearity after application of 25 mg, 50 mg and 100 mg Valoron N solution, b) dose equivalence of Valoron N solution (4 x 50 mg tilidine) and Valoron N retard tablets (2 x 100 mg tilidine) under steady state conditions, and c) the equivalence of different dose strengths of Valoron N retard tablets (50 mg, 100 mg, 200 mg tilidine/tablet). The results obtained in these studies demonstrate a dose linear kinetic for nortilidine after the application of 25 mg to 100 mg tilidine. Furthermore, there is dose equivalence between the tilidine/naloxone solution and tilidine/naloxone retard tablets, which permits the replacing of the solution with the retard tablets. Because of the equivalence of different dose strengths of Valoron N tablets, patients are able to exchange low dosed Valoron N retard tablets for higher-dosed ones (50 mg, 100 mg and 200 mg tilidine/tablet), if necessary. With their constant release of tilidine and the possibility for individual dosage, the retard tablets are efficient analgesics that improve pain therapy considerably for patients with chronic pain.

Tilidine does not affect human sphincter of Oddi motility--a randomized, controlled study.

AIM: To investigate the effects of intravenous pentazocine and tilidine on sphincter of Oddi motility. METHODS: Twenty patients with suspected sphincter of Oddi dysfunction were enrolled in a prospective, double-blind study. Sphincter of Oddi motility was assessed by means of endoscopic manometry after injection of 0.9% saline, as well as after randomized dosing with either 30 mg pentazocine i.v. (n = 10) or 50 mg tilidine i.v. (n = 10). RESULTS: Pentazocine significantly increased the sphincter of Oddi baseline pressure from 32 +/- 21 mmHg (saline) to 41 +/- 19 mmHg (P = 0.002), whereas tilidine did not alter the sphincter baseline pressure (34 +/- 15 mmHg saline vs. 36 +/- 16 mmHg tilidine, P = 0.16). Furthermore, pentazocine increased the phasic sphincter contraction amplitude (108 +/- 16 mmHg saline vs. 121 +/- 18 mmHg pentazocine, P = 0.004), but tilidine was without any effect (125 +/- 24 mmHg saline vs. 125 +/- 21 mmHg tilidine, P = 0.93). The phasic sphincter of Oddi contraction frequency and duration were not influenced either by pentazocine or by tilidine. CONCLUSION: In contrast to 30 mg of pentazocine, 50 mg of tilidine does not affect sphincter of Oddi motility. Therefore, tilidine can be used during endoscopic manometry and for analgesia in pancreatobiliary disease.

Dacarbazine and interferon alpha for stage IV malignant melanoma.

Based on encouraging reports of improved response rates with the use of dacarbazine (DTIC) in combination with recombinant interferon alpha-2a (rIFN-alpha-2a) in patients with metastatic malignant melanoma, we conducted a phase II study to determine the efficacy and feasibility of this treatment regimen. 31 patients were treated with an induction dose of rIFN-alpha-2a at 15 MIU/ m2 intravenously (i.v.) daily for 5 days per week for 3 consecutive weeks followed by a continuous maintenance dose of 10 MIU/m2 subcutaneously (s.c.) given 3 days per week; starting on day 22, in conjunction with rIFN-alpha-2a s.c., DTIC was started at a dose of 200 mg/m2 i.v. for 5 continuous days completing a 28-day cycle. Therapy was continued until progression was evidenced. Of the 29 evaluable patients, 7 (24.1%) achieved an objective response (complete plus partial remission) with the highest responses occurring in those patients assessed with pulmonary metastases. The median duration to treatment failure was 2.6 months, while the median survival was 6.9 months. Our data reveal that using rIFN-alpha-2a plus DTIC in combination does not yield better results than those achieved when using DTIC alone. However, 3 of the 7 responders experienced long-term survival ranging up to 42 months. Whether this benefit is achieved by the addition of rIFN-alpha-2a can only be answered by large randomized clinical trials. Conflicting results with some of the current literature are discussed.

Clinical and immunomodulatory effects of repetitive 2-day cycles of high-dose continuous infusion IL-2.

High-dose interleukin-2 (IL-2) treatment has demonstrated promising antitumour activity in renal cell carcinoma (RCC) and malignant melanoma (MM) and has been shown to induce broad immunological effects. The optimal IL-2 dose and schedule, however, still remain to be defined. We studied a treatment protocol consisting of five repetitive cycles of high-dose recombinant (rh) IL-2 (24 x 10(6) U/m2/day) administered weekly on two consecutive days by continuous intravenous infusion. 17/19 were RCC patients, 2 of whom responded with a complete remission (CR) and 3 with a partial response (PR) (CR + PR: 29%; median response duration of 11.5+ months (range: 3-14 months)). IL-2 induced a pronounced increase of lymphocytes and pro-inflammatory cytokines IL-8, IL-5, gamma-IFN, TNF- alpha and TNF-beta (p < 0.05) that peaked in cycle 3. With subsequent therapy, serum levels of these cytokines, NK, T cells and eosinophils decreased, whereas serum IL-10 levels progressively increased with maximum levels achieved after the fifth week of treatment, suggesting that it may be involved in dampening the inflammatory response induced by IL-2. Absolute numbers of activated T cells and NK cells remained elevated as compared to baseline for at least 4 weeks after treatment cessation. Based on these observations, future scheduling of IL-2 will be done at 3 weekly 2-day cycles separated by a week 4 treatment-free interval in order to increase further the 29% objective response rate achieved in this study.

Induction of tumor-specific cytotoxic T lymphocytes by immunization with autologous tumor cells and interleukin-2 gene transfected fibroblasts.

In a phase I trial designed to study a vaccine composed of autologous tumor cells and interleukin-2 gene transfected fibroblasts we analyzed lymphocytes infiltrating the vaccination site (VIL) in two melanoma patients. Functional studies demonstrated that numbers of MHC class I restricted cytotoxic T cells directed against the autologous tumor had increased at the immunization site in both cases. Analysis of the variability of T cell receptors (TCR) in the VIL of one patient revealed that the cytotoxic T lymphocytes consisted of a predominant population of TCRBV21S3+ T cells. Enrichment of this subpopulation to more than 99% by specific anti-TCRBV21S3 monoclonal antibody linked immunomagnetic beads and sequencing of the TCR-beta chain disclosed exactly the same V-D-J junctional sequence in all eight TCRBV21 transcripts from these VIL. The identical sequence was also detected in all eight TCRBV21 transcripts from the patient's tumor-infiltrating lymphocytes, indicating that the same CTL clone had infiltrated the tumor, circulated in the peripheral blood, and was amplified at the vaccination site. The TCRBV21S3+ T cells were also found to display an MHC class I restricted cytotoxic activity specifically directed against the autologous tumor cells. At the beginning of treatment these cells were undetectable at the vaccination site and delayed-type hypersensitivity testing was negative, contrasting with the positive results after therapy. Thus it is likely that vaccination with autologous tumor cells plus interleukin-2 gene transfected allogeneic fibroblasts had induced not only local accumulation but also an increase in the frequency of circulating tumor specific CTL.

A phase-I clinical study of autologous tumor cells plus interleukin-2-gene-transfected allogeneic fibroblasts as a vaccine in patients with cancer.

Tumor cells transfected to express immunostimulatory cytokines, or admixed with similarly modified bystander cells, are able to induce immune responses against unmodified tumor cells in animal models. For treatment of human patients, a vaccine composed of autologous tumor cells and IL-2-secreting allogeneic fibroblasts was developed. Autologous tumor cells were isolated from biopsy specimens. A clone (KMST 6.14) of an immortalized human fibroblast line that stably secreted 5290 IU IL-2 per 10(6) cells and per 24 hr was obtained by cationic lipofection with an expression construct for human IL-2 and Neo(r). Fifteen patients with refractory malignant tumors received 3-4 injections of irradiated KMST6.14 and autologous tumor cells in a phase-I clinical trial. Increasing transient inflammatory responses without systemic toxicity developed at vaccination sites and after injections with irradiated tumor cells only (p < 0.05). These sites contained a dense infiltrate of CD3+ T cells with numbers of CD4+ helper cells exceeding those of CD8+ cytotoxic T cells (CTL). CD8+ T-cell lines isolated from vaccination sites of 2 malignant melanoma patients but not of renal-cell carcinoma patients exhibited a dominant lytic activity against autologous tumor cells in vitro. CD8+ T-cell clones established from the vaccination site of 1 of 2 renal-cell carcinoma patients preferentially lysed autologous and partially matched allogeneic renal-cell carcinoma cells. In conclusion, a vaccine composed of IL-2 gene-transfected allogeneic fibroblasts and autologous tumor cells is able to enhance specific anti-tumor T-cell responses in vivo without major side-effects. Malignant melanoma and renal-cell carcinoma appear to be promising entities for testing of similar approaches in future therapeutic trials.

Processing of tumor tissues for vaccination with autologous tumor cells.

Vaccination with gene-transfected tumor cells has recently been proposed as a new strategy in the immunotherapy of cancer. Since autologous tumor cells provide an optimal antigen profile, the possibility of generating single cell suspensions from renal cell carcinoma (RCC), malignant melanoma (MM), colon carcinoma (CC), and non-small-cell lung cancer (NSCLC) biopsies was investigated. One hundred and seventy-four tumor biopsies were processed by mechanic and enzymatic dissociation, yielding 1-2 x 10(6) cells/g tumor (median), irrespective of tumor type. Primary tumor cell cultures (PTCC) of > or = 10(7) cells were established from 29 of 86 (34%) RCC, 14 of 38 (37%) MM, 11 of 23 (48%) NSCLC and 4 of 27 (15%) CC specimens. The amount of non-tumor cells, as assessed by morphology and immunocytology, was generally low (< 30%) in RCC (35 of 41) and MM (11 of 17), while it exceeded 60% in 8 of 11 PTCC from NSCLC and 3 of 11 CC. A high tumor cell yield was obtained in biopsies with a high degree of vascularization and in the virtual absence of necrosis. Thus, PTCC > or = 10(7) cells were obtained in 73% of MM with a high degree of vascularization and in 22% of MM with a low degree of vascularization (p < 0.007). Long-term tumor cell cultures exceeding 20 passages were established in 24 of 86 (18%) RCC, 7 of 38 (18%) MM and 3 of 27 (11%) CC, while successful implantation in nude mice was achieved in 8 of 20 RCC and 5 of 10 MM. Thus, under the conditions described, > or = 10(7) primary tumor cells of high purity could be generated from about one third of RCC and MM biopsies, while the success rate increased to > 50 and > 70%, respectively, in samples with a high degree of vascularization generated by an optimized biopsy technique excluding necrotic parts.

Mechanisms of p53 alteration in acute leukemias.

Disruption of normal p53 expression is the most frequent genetic change occurring in various human solid tumors; it is mostly due to sequence alterations of the p53 coding region by missense mutations or to loss of an entire, functional allele of this gene. In the present study, possible mechanisms resulting in a disruption of regulated expression of wild-type p53 were examined in acute leukemias of either lymphoid (ALL) or myeloid (AML) phenotype. p53 transcript accumulation, nucleotide sequence and gene structure were analyzed in primary leukemic cells from 50 patients. p53-specific transcripts were detected in 26/26 cases of ALL and 16/23 cases of AML using reverse transcriptase (RT)-PCR. Sequencing of transcripts did not reveal any point mutations or deletions. Heterozygosity at a polymorphic Bg/II site within intron 1 was found in 4/28 leukemic samples, and loss of one allele was noted in one of these. In addition, a novel, leukemia-associated structural abnormality located within the 5' flanking region of the p53 gene and associated with the loss of heterozygosity was observed in cells from this patient with ALL. The MDM2 gene which inactivates p53 by binding to it was neither amplified nor rearranged in 28 leukemias studied. Thus, disruption of regulated p53 expression resulting in lack of detectable p53 mRNA even by RT-PCR occurs in about 30% of cases of AML; however, p53 alterations typical for human solid tumors are an infrequent event in most types of human acute leukemias.

Burkitt-like mutations in the c-myc gene locus in prolymphocytic leukemia.

The c-myc oncogene recently shown to act as a transcription factor, is involved in cellular proliferation. Deregulation of this gene can be one step in malignant transformation. In Burkitt's lymphoma (BL) the c-myc gene is consistently involved in chromosomal translocations and the first exon of the gene has been found to be a frequent target of somatic mutations. These mutations are believed to interfere with normal transcriptional regulation of the gene. We demonstrate a case of the rare prolymphocytic leukemia (PLL), a variant of chronic lymphocytic leukemia (CLL), that shows multiple Burkitt-like mutations in the first exon of c-myc and one nonconservative point mutation in the coding exon 2. Cytogenetic analysis revealed involvement of both chromosomes 8 in chromosomal translocations. Both chromosomes 8 are broken at (q23), the c-myc gene locus. Since the patient's leukemia cells exhibited high expression levels of the mutated allele of the c-myc mRNA, the point mutations alone may have accounted for transcriptional deregulation.

Raf-1 is a necessary component of the mitogenic response of the human megakaryoblastic leukemia cell line MO7 to human stem cell factor, granulocyte-macrophage colony-stimulating factor, interleukin 3, and interleukin 9.

We have examined the role of Raf-1 in the mitogenic response of the factor-deprived human megakaryoblastic leukemia cell line MO7 to recombinant human granulocyte-macrophage colony-stimulating factor, interleukin 3, interleukin 9, and stem cell factor by using c-raf antisense oligodeoxyribonucleotides. Uptake of oligodeoxyribonucleotides by MO7 cells was maximal at 5-10 h in culture, and oligomers remained stable in these cells for at least 24 h. Treatment of MO7 cells with the antisense oligomer resulted in intracellular oligomer/mRNA duplex formation followed by efficient translation blockade of c-raf-1. In contrast, sense and non-sense oligodeoxyribonucleotides failed to form intracellular duplexes and did not interfere with translation of c-raf-1, suggesting specific elimination of c-raf-1 by the antisense oligodeoxyribonucleotide. Furthermore, exposure of MO7 cells to c-raf-1 antisense prevented factor-induced nuclear translocation of Raf-1. Most importantly, proliferation of MO7 cells ([3H]thymidine incorporation) enabled by these growth factors was significantly reduced when the c-raf-1 antisense oligodeoxyribonucleotide was added to cultures, whereas the mitogenic response to these factors remained almost unaffected in the presence of sense and non-sense oligodeoxyribonucleotides.

The complete sequence and promoter activity of the human A-raf-1 gene (ARAF1).

The raf proto-oncogenes encode cytoplasmic protein serine/threonine kinases, which play a critical role in cell growth and development. One of these, A-raf-1 (human gene symbol, ARAF1), which is predominantly expressed in mouse urogenital tissues, has been mapped to an evolutionarily conserved linkage group composed of ARAF1, SYN1, TIMP, and properdin located at human chromosome Xp11.2. We have isolated human genomic DNA clones containing the expressed gene (ARAF1) on the X chromosome and a pseudogene (ARAF2) on chromosome 7p12-q11.21. Analysis of the nucleotide sequence from the ARAF1 genomic clones demonstrated that it consists of 16 exons encoded by minimally 10,776 nucleotides. The major transcriptional start site (+1) was determined by RNase protection and primer extension assays. Promoter activity was confirmed by functional assays using DNA fragments fused to a CAT reporter gene. The ARAF1 minimal promoter, located between nucleotides -59 and +93, has a low G + C content and lacks consensus TATA and Inr sequences but shows sequence similarity at position -1 to the E box that is known to interact with USF and TFII-I transcription factors.

The mitogenic response of T cells to interleukin-2 requires Raf-1.

The product of the c-raf-1 proto-oncogene, Raf-1, is known to encode a 74-kDa ubiquitously expressed cytoplasmic serine/threonine kinase. Various growth factors such as epidermal growth factor, acidic fibroblast growth factor, platelet-derived growth factor, insulin, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-2, IL-3 and erythropoietin have been shown to induce phosphorylation of Raf-1, thereby activating Raf-1 kinase. Raf-1 is, thus, believed to play a role in coupling growth factor receptors to proliferation. We have examined the role of Raf-1 in the mitogenic response of human peripheral blood-derived IL-2 receptor expressing T cells to human recombinant IL-2 employing c-raf antisense (AS) oligodeoxyribonucleotide. Uptake studies of oligonucleotides indicated that incorporation of oligomers was maximal at 4 h and oligodeoxynucleotides remained stable in these cells for up to 24 h. Treatment of T cells with the AS oligodeoxyribonucleotide in intracellular duplex formation followed by efficient translation blockade of c-raf-1. In contrast, sense (S) and nonsense (NS) oligodeoxynucleotides failed to form intracellular duplexes and did not interfere with translation of c-raf-1, suggesting specific elimination of c-raf-1 by the AS oligomer. Proliferation of T cells ([3H]thymidine incorporation) following exposure to IL-2 was substantially reduced when the c-raf-1 AS oligodeoxyribonucleotide was added to cultures, while the mitogenic response to this factor remained almost unaffected in the presence of S and NS oligodeoxyribonucleotides.

Induction of c-jun expression in the myeloid leukemia cell line KG-1 by 1-beta-D-arabinofuranosylcytosine.

c-Jun/AP-1 is a transcription factor commonly induced in mammalian cells by serum, phorbol compounds, or peptide growth factors. We show that c-Jun/AP-1 is inducible as well as coordinately regulated, in the human acute myelogenous leukemia cell line KG-1, by the cytostatic drug 1-beta-D-arabinofuranosylcytosine (Ara-C). Concomitantly with Ara-C treatment, growth inhibition and loss of clonogenic survival of KG-1 cells were observed. Whereas KG-1 cells displayed only barely detectable amounts of c-jun transcripts when cultured in the presence of serum, Ara-C at concentrations of 1 to 50 microM induced c-jun transcripts in a dose-dependent fashion. Time course studies showed that 10 microM Ara-C induced c-jun transcripts 6 hr after initiation of culture. Induction of c-jun mRNA was independent of de novo protein synthesis, because the protein synthesis inhibitor cycloheximide failed to alter Ara-C-induced c-jun mRNA accumulation. Furthermore, cycloheximide did not induce c-jun transcripts, ruling out the possibility of posttranscriptional stabilization of c-jun mRNA by labile proteins, as has been previously reported for a variety of serum-inducible protooncogenes and early response genes. Moreover, nuclear run-on analysis disclosed that c-jun induction by Ara-C in KG-1 cells took place at a transcriptional level. Taken together, these findings indicate that c-jun mRNA, unlike its rapid (within minutes) induction by serum in fibroblasts, is induced by Ara-C in KG-1 cells following a much more prolonged time course and is regulated essentially at a transcriptional level.