Simultaneous determination of gemcitabine di- and triphosphate in human blood mononuclear and cancer cells by RP-HPLC and UV detection.

A reverse-phase HPLC method based on ion-pair formation with UV detection was set up for the simultaneous determination of gemcitabine diphosphate (dFdCDP) and triphosphate (dFdCTP) in human cells. The separation was achieved on a Tracer Excel ODSA column (100 mm x 4.6mm i.d., 3 microm particle size) at room temperature. Nine nucleotides were separated by isocratic elution in 26 min. Accuracy, linearity, sensitivity and precision studies for dFdCDP, dFdCTP, adenosine diphosphate (ADP) and triphosphate (ATP) validated this method. This assay was used to provide data from gemcitabine treated patients and in vitro grown human cancer cells.

Down-regulation of the beta-chemokine receptor CCR6 in dendritic cells mediated by TNF-alpha and IL-4.

Chemokines are involved in the control of dendritic cell (DC) trafficking, which is critical for the immune response. We have generated DC from human umbilical cord blood CD34+ progenitors cultured with granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha (TNF-alpha), and stem cell factor. Using an anti-CCR6 monoclonal antibody, we observed that these cells showed maximum expression of this beta-chemokine receptor when they were immature, as determined by their relatively low expression of several DC maturation markers such as CD1a, CD11c, CD14, CD40, CD80, and CD83. Immature DC responded strongly to macrophage inflammatory protein-3alpha (MIP-3alpha), the CCR6 ligand, in migration and calcium mobilization assays. CCR6 expression decreased in parallel with the DC maturation induced by prolonged TNF-alphaq treatments. Interleukin-4 was also able to decrease CCR6 protein levels. Our findings suggest that the MIP-3alpha/CCR6 interaction plays an important role in the trafficking of immature DC to chemokine production sites such as injured or inflamed peripheral tissues, where DC undergo maturation on contact with antigens.

Determination of thiamine and its phosphorylated forms in human plasma, erythrocytes and urine by HPLC and fluorescence detection: a preliminary study on cancer patients.

In man, neurotoxicity associated to ifosfamide treatment can be reversed by intravenous thiamine administration. Trying to explain this clinical finding, we decided to study possible changes in thiamine availability and activation in patients exposed to ifosfamide. Free thiamine and its phosphate esters levels were measured in plasma, erythrocytes and urine by an ion-pair HPLC method with pre-column derivatization, which allowed separation of the fluorescent compounds in less than 10 min. The method was validated by linearity, sensitivity and reproducibility studies, whose values met the demands for bioanalytical assays. This method was applied to assess thiamine status in cancer patients exposed to ifosfamide therapy for advanced disease.

Complete nucleotide sequence of the penicillin acylase gene from Kluyvera citrophila.

The penicillin acylase (PAC) from Kluyvera citrophila ATCC21285 has been purified to homogeneity and found to be composed of two non-identical subunits of 23 and 62 kDa, in contrast with the previous findings [Shimizu et al., Agr. Biol. Chem. 39 (1975) 1655-1661]. The nucleotide (nt) sequence of the K. citrophila pac gene contained in the 3-kb PvuI-HindIII fragment of pKAP1 [García and Buesa, J. Biotechnol. 3 (1986) 187-195] has been determined, showing that it encodes a protein of 844 amino acid (aa) residues. The aa analysis of the N-terminal and C-terminal sequences of the purified subunits showed that they were derived from a common precursor protein of 93.5 kDa, from which a signal peptide of 26 aa, responsible for the periplasmic translocation of the protein, and an internal connecting polypeptide of 54 aa, have been removed in the maturation of the PAC. The comparison of the nt sequences of the pac genes from K. citrophila and Escherichia coli ATCC11105 [Schumacher et al., Nucl. Acids Res. 14 (1986) 5713-5727] revealed 80% homology, suggesting a common ancestral pac gene origin. The results reported here should allow investigation of the unusual mechanism of maturation of this prokaryotic protein, as well as manipulation, using DNA recombinant techniques, of the catalytic properties of this industrially important enzyme.

Temozolomide in adult patients with advanced soft tissue sarcoma: a phase II study of the EORTC Soft Tissue and Bone Sarcoma Group.

Temozolomide, an oral imidazotetrazine derivative, was given to 31 patients with advanced soft tissue sarcoma. The dose of 750 mg/m2 was divided over 5 consecutive days, and escalated to 1000 mg/m2 over 5 days at cycle 2 if myelosuppression no worse than common toxicity criteria grade 2 was noted in the first 28-day cycle. A total of 99 treatment cycles were given to 31 patients. The drug was well tolerated, with nausea and vomiting as the most common side-effects. Only one partial tumour response was documented, giving a response rate of 3.33%, 95% confidence interval, (CI) 0.1-17.2%. The median time to progression was 8 weeks and the median survival was 27 weeks. These results indicate that temozolomide in this schedule is not active as second-line treatment in advanced soft tissue sarcoma.

Molecular cloning and expression of stromalin protein from Drosophila melanogaster: homologous to mammalian stromalin family of nuclear proteins.

We report the cloning of a new cDNA from Drosophila melanogaster that encodes an open reading frame of 1116 amino acid residues. It is the insect homolog of the previously reported stromalin (SA) family of nuclear proteins in mammals (Carramolino et al. [1997]. Gene 195, 151-159). Taking into account the identical domain present in all the SA family members characterized to date, we have carried out polymerase chain reaction (PCR) using degenerate oligonucleotides from the 5' and 3' ends of one of those regions of the molecule and cDNA from D. melanogaster embryos. We isolated the homologous domain of the putative Drosophila SA molecule (DSA). This cDNA fragment was used as a radiolabeled probe for screening a cDNA library from Drosophila embryos, and we have cloned a full-length cDNA for the SA homolog from an insect. The protein shows a good degree of identity with the mammalian stromalins SA-1 and SA-2, with the N and C ends being the most divergent regions of the molecule. The mRNA coding for this protein shows a molecular size of about 3.7 kb by Northern blot analysis and is essentially expressed in embryonic stage. The in situ hybridization experiments indicate that the DSA messenger is expressed mainly in neurogenic territories in the embryonic development of Drosophila. The DSA protein has been cloned and expressed in a baculovirus system, and polyclonal antibodies were generated against the recombinant molecule. Western blot analysis using these antibodies detected a main band corresponding to about 120 kDa, principally in embryos.

Clinical pharmacokinetics of high-dose DTIC.

The pharmacokinetics of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC, dacarbazine) given at a dose of 850-1,980 mg/m2 as a 10- to 30-min infusion was studied in cancer patients, and the plasma concentration-time curves were adjusted to a two-compartment model, with a mean t1/2 alpha value of 0.17 h (range, 0.1-0.26 h) and a mean t1/2 beta value of 2 h (range, 1.5-2.7 h) being found. The mean volume of the central compartment of (Vc) and the apparent volume of distribution (VB) were 0.42 1 kg-1 (range, 0.24-0.54 1 kg-1) and 1.49 1 kg-1 (range, 0.88-1.74 1 kg-1), respectively. The mean total body clearance of DTIC was 0.58 1 kg-1 h-1 (range, 0.26-0.82 1 kg-1 h-1), and the mean renal clearance was 0.28 1 kg-1 h-1 (range, 0.17-0.49 1 kg-1 h-1). Unchanged DTIC recovered from urine within 24 h varied from 11% to 63% of the delivered dose, with an inverse correlation being found between the DTIC dose and the amount excreted. The metabolite aminoimidazole carboxamide (AICA) was detectable in plasma from the start of DTIC infusion, and its concentration-time curve showed a monophasic decay, exhibiting a mean t1/2 value of 3.25 h (range, 1.77-5.82 h). Mean AICA renal clearance was 0.15 1 kg-1 h-1 (range, 0.05-0.32 1 kg-1 h-1). The amount of AICA excreted in urine increased with increasing DTIC dose and varied from 1.2% to 13.6% of the delivered DTIC dose. Both DTIC distribution and disposition and AICA production and renal excretion seemed to be limited after high DTIC doses as compared with the pharmacokinetics of low-dose DTIC. Nonlinear pharmacokinetics for high-dose DTIC could not be clearly excluded.

Phase II clinical trial of cis-dichlorodiammineplatinum in gastric cancer.

Eighteen patients with gastric cancer in Stage IV who had failed combination chemotherapy were treated with cis-dichlorodiammineplatinum as a single agent. Objective response was observed in four patients (22%), and another two had stable disease lasting 114 and 234 days, respectively. The median duration of the response was 150 days (range, 92-186). Gastrointestinal toxicity occurred in all the patients, but leukopenia and thrombocytopenia was the major complication in patients who had been heavily pretreated. The response rate found in this study shows that cis-platinum is an active drug in gastric cancer.

Phase II Trial of Doxorubicin Plus Escalated High-Dose Ifosfamide in Patients With Advanced Soft Tissue Sarcomas of the Adult: A Study of the Spanish Group for Research on Sarcomas (GEIS).

Background. To explore the tolerance and the activity of high-dose ifosfamide (IFOS) combined with doxorubicin (DXR) at 50 mg/m(2) every 4 weeks in patients with soft tissue sarcomas. Methods. DXR was given IV bolus and IFOS by continuous infusion at 2 g/m(2)/day. Initial IFOS dose (12 g/m(2)) was adjusted to 10, 13, or 14 g/m(2) according to toxicity. Results. Seventy patients received 277 cycles (median 3 cycles, range 1-10), 34% with IFOS dose increased, 30% decreased, and 48% delivered at 12 g/m(2). Toxicity grade 4 occurred on granulocytes (67% of patients) or platelets (19%), 54% had febrile neutropenia, 31% grade 3/4 asthenia, and 26% abandoned the study due to toxicity. Three toxic deaths occurred. In 57 non-GIST patients objective activity was 45.6% (95% CI, 32 to 58%). Conclusion. At least 4 cycles were tolerated by 71% of patients, most receiving DXR 50 mg/m(2) plus IFOS 10-12 g/m(2), with substantial toxicity.

Phase I/II trial of doxorubicin and fixed dose-rate infusion gemcitabine in advanced soft tissue sarcomas: a GEIS study.

The aim of the study was to determine the dose-limiting toxicity and maximum tolerated dose of a first-line combination of doxorubicin and gemcitabine in adult patients with advanced soft tissue sarcomas and to explore its activity and toxicity, and the presence of possible interactions between these agents. Patients with measurable disease were initially treated with doxorubicin 60 mg m(-2) by i.v. bolus on day 1 followed by gemcitabine at 800 mg m(-2) over 80 min on days 1 and 8, every 21 days. Concentrations of gemcitabine and 2',2'-difluorodeoxyuridine in plasma, and gemcitabine triphosphate levels in peripheral blood mononuclear cells were determined during 8 h after the start of gemcitabine infusion. Myelosuppression and stomatitis were limiting toxicities, and the initial dose level was applied for the Phase II trial, where grade 3-4 granulocytopenia occurred in 70% of patients, grade 3 stomatitis in 46% and febrile neutropenia in 20%. Objective activity in 36 patients was 22% (95% CI: 9-35%), and a 50% remission rate was noted in leiomyosarcomas. Administration of doxorubicin preceding gemcitabine significantly reduced the synthesis of gemcitabine triphosphate. Clinical activity, similar to that of single-agent doxorubicin, and the toxicity encountered do not justify further studies with this schedule of administration.

Phase II Clinical Trial With Pegylated Liposomal Doxorubicin (CAELYX(R)/Doxil(R)) and Quality of Life Evaluation (EORTC QLQ-C30) in Adult Patients With Advanced Soft Tissue Sarcomas: A study of the Spanish Group for Research in Sarcomas (GEIS).

BACKGROUND: Pegylated liposomal doxorubicin (PLD), a formulation with pharmacokinetic differences with respect to doxorubicin (DXR), might benefit patients with advanced soft tissue sarcoma (STS) pretreated with DXR. PATIENTS AND METHODS: Patients with measurable and progressive STS received PLD at 35 mg/(2) every 3 weeks. Quality of life before and during treatment was assessed with EORTC QLQ-C30. RESULTS: Twenty-eight patients, 22 DXR-pretreated, were given 140 cycles (median 3, range 1-18). Activity in 27 patients (5 GIST): one complete and one partial remission (both non-GIST and without prior DXR), 12 stabilizations and 13 progressions (response rate 7.4%, 95% CI: 0-17%). Grade 3 toxicity: palmar-plantar erythrodysesthesia (19% of patients), stomatitis (4%) or cutaneous (4%). Neutropenia grade>/=3 was detected in 16% of patients. Median relative dose intensity was 95%. Progression-free rate at 3 and 6 months was, respectively, 48 and 22%, median progression-free survival 5.8 months and median overall survival 8.7 months. QLQ-C30 at baseline and at weeks 6-11 in 23 and 13 patients, respectively, showed good reliability and validity. Quality of life did not seem to worsen during therapy. CONCLUSIONS: PLD did not induce objective remissions in 22 STS patients pretreated with DXR, but progression-free rate figures support the use of this agent in patients who have not progressed under a DXR-containing regimen. The toxicity observed was comparable to that of other PLD schedules.

[Use of purine rescue pathways by L1210 cells exposed to methotrexate in vitro]. / Utilizacíon de las vías de rescate de purinas por celulas L1210 expuestas a methotrexate (MTX) in vitro.

Methotrexate (MTX) blocks the de novo synthesis of purines and pyrimidines due to a diminution of reduced folates, and produces an accumulation of intracellular 5-phosphoribosyl-1-pyrophosphate (PRPP). Phosphoribosyltransferases, in the presence of PRPP, synthesize nucleotides starting on purine bases, that are activated to triphosphates ("rescue pathway"). The authors study the activity of the rescue pathways in L1210 leukemia cells in vitro to restore the levels of ATP and GTP that have been lowered by MTX. Hypoxanthine (Hpx) was used as a preformed source of purines, high efficiency liquid chromatography was employed to estimate the intracellular concentrations of ATP and GTP. Two hours after 0.1 mM Hpx addition to in vitro L1210 cells in the presence of 0.1 microM MTX, the amount of ATP and GTP increased 5 to 7 times. In former experiments the author has shown that the uptake of PRPP increases when Hpx is added to L1210 cells exposed to MTX. The results of this work show that L1210 leukemia cells in vitro can use the rescue pathways of purine synthesis when Hpx is present in the growth medium, so preventing the anti-purine effect of MTX.

Phase I and II clinical study of anhydro-ara-5-fluorocytosine (AAFC) and ICRF-159 combination in adenocarcinoma of digestive origin.

The results of a phase I--II study of a combination chemotherapy with AAFC and ICRF-159 in advanced adenocarcinoma of digestive origin are presented. Myelosuppression was the dose-limiting toxicity with anemia, leukopenia, and thrombocytopenia. The maximum tolerated dose of AAFC in the combination program was 650 mg/m2 I.V. weekly. ICRF-159 was given in a 3-day course every 3 weeks and the dose was escalated from 125 mg/m2 to 500 mg/m2 daily. Bone marrow toxicity was noticied at the first escalation level and all dose levels were similarly toxic. The results of this combination chemotherapy were: two partial responses in 14 patients with gastric cancer; no responses in nine patients with colorectal cancer; no responses in three patients with pancreatic cancer; and no responses in two patients with biliary tree cancer. In conclusion, AAFC and ICRF-159 combination chemotherapy demonstrated a low level of activity in advanced carcinoma of digestive origin. The peculiar hematologic toxicity found at the low-level dose requires further documentation and could make this drug association suitable for a phase II study in leukemia and/or lymphoma.