Ecomustine, or CY233 (NSC-609224), is a new water-soluble nitrosoureido sugar derived from acosamine. A high-performance liquid chromatographic assay (HPLC) developed to quantify the unchanged drug in aqueous solutions and biological specimens enabled us to study the chemical stability as a function of pH, light, and temperature. In buffered aqueous solutions, the kinetics of degradation of CY233 is a first-order process. The log k-pH profile demonstrated hydroxide ion-catalyzed solvolysis. The drug is most stable at pH 4, more stable than some other nitrosoureas in 5% glucose (t1/2, 62-67 h) and in 0.9% isotonic saline (t1/2, 25-37 h) solutions. Based on these findings, blood samples should be collected in cold tubes (4 degrees C) containing citrate buffer (pH 4) and all manipulations should be protected from heat and light.

The aim of this study was to compare intra-arterial hepatic administration (IAH) versus i.v. administration of oxaliplatin and cisplatin in a VX2 tumor model in rabbits. VX2 tumors were implanted in the livers of White New Zealand female rabbits and 2 weeks later they received either cisplatin (4 mg/kg) or oxaliplatin (6 mg/kg) administered by IAH or i.v. Platinum pharmacokinetic parameters were measured by atomic absorption spectrometry at baseline, 2, 5 10, 20, 40 and 60 min, and then at 2, 4, 6 and 24 h after drug administration. Animals were sacrificed 24 h after drug administration to measure platinum concentrations in various tissues. After IAH oxaliplatin administration, we observed a significant decrease for total and filterable platinum in the Cmax compared with i.v. administration (12.4 versus 18.2 microg/l; p=0.02 and 11.2 versus 17.3 microg/l; p=0.02, respectively). Significant differences in various tissue concentrations were reported when comparing IAH and i.v. administration of oxaliplatin with IAH administration offering an advantage over i.v. administration. No differences in pharmacokinetic parameters or platinum tissue accumulation were apparent between the IAH and i.v. administration with cisplatin. We conclude that there is a significant pharmacokinetic advantage to using oxaliplatin for locoregional IAH chemotherapy compared with i.v. administration.

Flavone acetic acid pharmacokinetics were studied in 31 patients in a phase I clinical trial. The drug was given by i.v. infusions over 1, 1.5, 3, and 6 h at doses ranging from 0.5 to 6.4 g/m2. The pharmacokinetic parameters were determined according to a nonlinear model including Michaelis-Menten-type kinetics. The mean elimination half-life is 4.8 h and the mean volume of distribution of the central compartment, 7.61. Our model predicted a maximal tolerated dose (MTD) of 11.1 g/m2 on the basis of the "therapeutic window" concept, very close to the clinically observed MTD of 10 g/m2. This model is also operational when different protocols of inoculation are considered, such as a divided-dose schedule vs a unique infusion, and indicates that, at the MTD, injections should be made every 72 h to avoid drug accumulation.

New nitrosoureido derivatives of di- or tri-deoxy-sugars have been synthesized. Very potent antitumour activity against L1210 leukaemia was exhibited by the compounds derived from methyl 3-amino-3,4-dideoxy-β- and α- and 4-amino-2,4-dideoxy-β- and α-D-arabino-hexopyranosides, 24, 26, 28 and 29, respectively. In further evaluation against B16 melanocarcinoma bearing mice, only compounds 24 and 26 displayed significant activity. Owing to its lower acute toxicity, methyl 3-[3-(2-chloroethyl)-3-nitrosoureido]-3,4-dideoxy-β-D-arabino-hexopyranoside 24 appeared as the best candidate for preclinical studies.

Several glycosyl derivatives of squamocin (1) have been synthesized by glycosylation under Lewis acid catalysis with two different 1-O-acetyl sugars. Separation of these compounds has been achieved by HPLC and centrifugal partition chromatography (CPC). A detailed NMR, ESIMS, and LSIMS study allowed complete structural elucidations. The cytotoxic activity of the glycosyl derivatives was investigated and compared with that of squamocin and dihydrosquamocin against human epidermoid carcinoma cells (KB), African green monkey (Cercopithecus aethiops) kidney epithelial cells (VERO), and mouse lymphocytic leukemia cells (L1210). The antiproliferative effects of some derivatives were studied on cell cycles in mouse lymphocytic leukemia cells (L1210).

Gene therapy using the herpes simplex virus thymidine kinase (HSV-TK) gene in combination with the drug ganciclovir (GCV) is a promising approach for the treatment of cancer-inducing gliomas, a tumor with a poor prognosis. In an attempt to limit the toxic effects on normal tissues, we constructed a recombinant adenoviral vector, Adgfa2TK, in which the HSV-TK gene is driven by the promoter for the gene encoding glial fibrillary acidic protein, an intermediate filament protein expressed primarily in astrocytes. Infection by Adgfa2TK of a glial cell line (C6) and a non-glial cell line (MDA-MB-231) revealed markedly increased expression of HSV-TK in glial cells as determined by Western blot. In comparison, high HSV-TK protein levels were produced in both cell lines after infection with a control virus, AdCMVTK, in which the constitutive cytomegalovirus viral promoter was used to direct HSV-TK expression. Infection of two glial cell lines (C6, U251) and two non-glial cell lines (HepG2, MDA-MB-231) with Adgfa2TK followed by GCV treatment revealed high toxicity in glial cell lines (50% growth inhibitory concentration: <2 μg/mL of GCV) with little or no toxicity (50% growth inhibitory concentration: >75 μg/mL) in the non-glial cell lines. In vivo, injection of Adgfa2TK into C6 tumors grown in nude mice followed by intraperitoneal GCV treatment significantly repressed tumor growth compared with the controls. Adgfa2TK may be useful for directing expression of the HSV-TK gene to gliomas.

BACKGROUND Cisplatin (cis-diamminedichloroplatinum) is one of the most active agents against a broad range of malignancies, including ovarian cancer. Cisplatin resistance appears to be associated with several molecular alterations, including overexpression of metallothionein, a metal-binding protein. In the present study, we attempted to take advantage of metallothionein overexpression to overcome cisplatin resistance. METHODS Using a virus-free system (liposomes), we sought to express the suicide gene, thymidine kinase (TK), driven by the promoter of the human metallothionein IIa (hMTIIa) gene using the pMT-TK plasmid. We used cisplatin-resistant human ovarian carcinoma cells as a model. RESULTS We first analyzed metallothionein expression using a ribonuclease protection assay. In comparison to parental cells, the cisplatin-resistant cells were found to have increased expression of metallothionein messenger RNA (mRNA). Metallothionein overexpression in these cells was not associated with an increased copy number of the hMTIIa gene or with different transfection efficiencies. Furthermore, we showed by reverse transcription-polymerase chain reaction analysis that transfection of the pMT-TK plasmid results in a 56-fold higher expression of thymidine kinase mRNA in cisplatin-resistant cells compared with parental cells, consistent with increased metallothionein promoter-mediated transactivation in the cisplatin-resistant cells. Transfection of resistant cells with pMT-TK or a control plasmid (pCD3-TK) resulted in a marked sensitization to ganciclovir, with a 50% cell growth-inhibitory concentration (IC(50)) of 20 microg/mL and 9 microg/mL, respectively. Transfections of the cisplatin-sensitive cells resulted in no sensitization to ganciclovir with pMT-TK (IC(50) 200 microg/mL) and a high sensitization with pCD3-TK (IC(50) = 6 microg/mL). CONCLUSION These studies suggest that pMT-TK gene therapy may provide an alternative treatment for cisplatin-refractory ovarian tumors.

7-Ethyl-10[4-(1-piperidino)-1-piperidino] carbonyloxy-camptothecin (CPT-11), a DNA topoisomerase I inhibitor, undergoes several metabolic pathways to generate conjugated and unconjugated derivatives that could be excreted from the body. The objective of this study was to determine the oxidative metabolites of CPT-11 recovered in human urine samples and to identify cytochrome P450 (CYP) involved in their formation. In addition to the already known metabolites of CPT-11 [SN-38, SN-38-G, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin (APC), and 7-ethyl-10-(4-amino-1-piperidino) carbonyloxycamptothecin (NPC)], we isolated three oxidized metabolites from the urine of two children and two adults given CPT-11. M1 and M2 (molecular weight, 602) were hydroxylated, respectively, on the CPT moiety and on the terminal piperidine ring of CPT-11. M3 had a molecular mass of 602, but its urine concentration in patients was too low to establish its chemical structure by liquid chromatography/mass spectrometry. In vitro incubations with cells expressing CYP2C8, CYP2C9, CYP1A1, CYP1A2, or CYP3A7 did not produce any detectable metabolites. Only CYP3A4 produced both APC and NPC, resulting from the oxidation of the piperidinylpiperidine side chain of CPT-11 along with metabolite M2. The metabolism of CPT-11 by CYP3A5 was markedly different because neither APC or NPC nor M2 was produced, whereas only one new metabolite, M4 (molecular weight, 558), was generated by de-ethylation of the CPT moiety. No previous study has reported the presence of the M4 metabolite. Production of APC, NPC, M2, and M4 was prevented by ketoconazole, a specific CYP3A inhibitor. The parameters of CPT-11 biotransformation into M2 and M4 were examined using cell lines expressing, respectively, with CYP3A4 and CYP3A5, indicating that CPT-11 is preferentially metabolized by CYP3A4. In conclusion, CYP3A plays a major role in the metabolism of CPT-11, with some differences of the metabolic profile exhibited by 3A4 and 3A5.

High-dose busulphan-containing chemotherapy regimens have shown high response rates in children with relapsed or refractory neuroblastoma, Ewing’s sarcoma and medulloblastoma. However, the anti-tumour activity of busulfan as a single agent remains to be defined, and this was evaluated in athymic mice bearing advanced stage subcutaneous paediatric solid tumour xenografts. Because busulphan is highly insoluble in water, the use of several vehicles for enteral and parenteral administration was first investigated in terms of pharmacokinetics and toxicity. The highest bioavailability was obtained with busulphan in DMSO administered i.p. When busulphan was suspended in carboxymethylcellulose and given orally or i.p., the bioavailability was poor. Then, in the therapeutic experiments, busulphan in DMSO was administered i.p. on days 0 and 4. At the maximum tolerated total dose (50 mg kg–1), busulphan induced a significant tumour growth delay, ranging from 12 to 34 days in the three neuroblastomas evaluated and in one out of three medulloblastomas. At a dose level above the maximum tolerated dose, busulphan induced complete and partial tumour regressions. Busulphan was inactive in a peripheral primitive neuroectodermal tumour (PNET) xenograft. When busulphan pharmacokinetics in mice and humans were considered, the estimated systemic exposure at the therapeutically active dose in mice (113 μg h ml–1) was close to the mean total systemic exposure in children receiving high-dose busulphan (102.4 μg h ml–1). In conclusion, busulphan displayed a significant anti-tumour activity in neuroblastoma and medulloblastoma xenografts at plasma drug concentrations which can be achieved clinically in children receiving high-dose busulphan-containing regimens.

Trecovirsen, a 25-mer antisense phosphorothioate oligonucleotide targeted at the gag site of the HIV gene, was administered to HIV-positive volunteers as an i.v. infusion. Single doses ranged from 0.1 to 2.5 mg/kg in an ascending escalation in cohorts of 6 to 12 subjects. Plasma trecovirsen concentrations and pharmacokinetic parameters could be assessed at doses > or = 0.3 mg/kg. Peak plasma concentrations and AUC values increased disproportionately with increasing dose while elimination half-life increased and plasma clearance decreased, indicating a saturable process over this dose range. The only significant adverse event observed was an isolated, transitory increase in activated partial thromboplastin time at doses > or = 2.0 mg/kg that was related to plasma trecovirsen concentrations and is attributed to the polyanionic character of the molecule. Thus, trecovirsen administration was well tolerated in single i.v. doses up to 2.5 mg/kg.

During inflammation and infection, overexpression of the tumor necrosis factor (TNF) is associated with changes in cytochromes P-450 levels in rat and human hepatocytes. The aim of this study was to investigate the effect of TNF on the expression of the glutathione-S-transferases (GSTs) in rat hepatocytes. TNF was added in vitro alone or simultaneously with phenobarbital (PB) into hepatocytes in primary culture or in vivo, before TNF, injected directly to rats. GST activity was assayed by spectrophotometry; protein GSTs alpha, mu and pi were evaluated by immunoblotting. When TNF was added alone to rat hepatocytes in vitro, total GST activity and GST alpha levels were not affected, while GST mu protein levels significantly decreased by 35%. GST pi protein was undetectable in hepatocytes whether treated or not with TNF. When PB was administered in vitro simultaneously to rat hepatocytes with TNF, the decrease observed for GST mu subunit was suppressed while total GST activity and GST alpha content were not affected. When hepatocytes were treated with TNF after PB given in vivo directly to the rat by i.p. injection, GST activity and GSTs subunits were induced by PB, while TNF did not exert any effect. These results indicate that TNF has an inhibitory effect on GST mu and PB abrogates this effect in primary cultured rat hepatocytes. Then, PB could prevent some TNF toxic effects.

Although the prognosis of childhood cancers has dramatically improved over the last three decades, new active drugs are needed. Camptothecins represent a very attractive new class of anticancer drugs to develop in paediatric oncology. The preclinical and clinical development of two of these DNA-topoisomerase I inhibitors, i.e. topotecan and irinotecan, is ongoing in paediatric malignancies. Here we review the currently available results of this evaluation. Topotecan proved to be active against several paediatric tumour xenografts. In paediatric phase I studies exploring several administration schedules, myelosuppression was dose-limiting. The preliminary results of topotecan evaluation in phase II study showed antitumour activity in neuroblastoma (response rate: 15% at relapse and 37% in newly diagnosed patients with disseminated disease) and in metastatic rhabdomyosarcoma (40% in untreated patients). Topotecan-containing drug combinations are currently investigated. Irinotecan displayed a broad spectrum of activity in paediatric solid tumour xenografts, including rhabdo-myosarcoma, neuroblastoma, peripheral primitive neuroectodermal tumour, medulloblastoma, ependymoma, malignant glioma and juvenile colon cancer. For several of these histology types, tumour-free survivors have been observed among animals bearing an advanced-stage tumour at time of treatment. The clinical evaluation of irinotecan in children is ongoing. Irinotecan undergoes a complex in vivo biotransformation involving several enzyme systems, such as carboxylesterase, UDPGT and cytochrome P450, in children as well as in adults. Preclinical studies of both drugs have shown that their activity was schedule-dependent. The optimal schedule of administration is an issue that needs to be addressed in children. In conclusion, the preliminary results of the paediatric evaluation of camptothecin derivatives show very encouraging results in childhood malignancies. The potential place of camptothecins in the treatment of paediatric malignant tumours is discussed.

Hypercholesterolemia increases the oxidation of low density lipoprotein (LDL) which subsequently leads to atherogenesis. The oxidized LDL are also known to increase in vitro macrophage synthesis of glutathione. The purpose of this study was to investigate the relationship between lipid parameters and the glutathione system (glutathione, glutathion S-transferase) in total blood and within leukocytes. The glutathione and glutathion S-transferase were evaluated by spectrophotometric methods in sixty-two healthy volunteers (32 women, 30 men, mean age 39.9 +/- 7.7). No correlation was found between the level of blood cholesterol and the values of the blood glutathione system. However, a positive correlation between the values of glutathione and glutathion S-transferase in leukocytes and the blood cholesterol level was only found in women (r = 0.55 and r = 0.50 respectively, p < 0.01). We also found in men a positive correlation between body mass index and glutathion S-transferase in total blood and within leukocytes (r = 0.38, p < 0.05, r = 0.5, p < 0.01 respectively). No correlation was found between age, smoking and the values of the glutathione system. Our results suggest that the glutathione system in leukocytes is related to blood cholesterol levels. The fact that this positive correlation was only observed in women points to a possible role of estrogens in the regulation of the glutathione system which merits to be further studied.

Gene therapy using the herpes simplex virus thymidine kinase gene (HSV-TK) is a promising new approach for the treatment of gliomas, a tumor type with a poor prognosis. To limit the toxic effects of this procedure, it is desirable to restrict expression of the HSV-TK gene to the target cells. This can be accomplished by use of the promoter of the glial fibrillary acidic protein gene, an intermediate filament protein expressed primarily in astrocytes. A plasmid containing the HSV-TK gene, driven by the human glial fibrillary acidic protein promoter gfa2, was lipofected into glioma cell lines and into an ovarian cancer cell line. Treatment with ganciclovir showed efficient killing of glioma cells, with no effect on the ovarian cells. Thus, the gfa2 promoter is a promising candidate for directing expression of toxic genes to gliomas.

Fotemustine is a chloroethylnitrosourea with antitumor activity in disseminated melanoma and adult primary brain tumors. Because new drugs are required for the treatment of medulloblastoma in children, we evaluated the preclinical antitumor activity of fotemustine in four s.c. medulloblastoma xenografts, in comparison with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Both drugs were administered as a single i.p. injection to nude mice bearing advanced-stage tumor. Fotemustine displayed significant antitumor activity in three of four medulloblastoma xenografts; two, IGRM34 and IGRM57, were highly sensitive, with 37 and 100% tumor-free survivors, respectively, more than 120 days after treatment at the highest nontoxic dose (50 mg/kg). Fotemustine was also highly active in a malignant glioma xenograft (IGRG88; five of six tumor-free survivors on day 177). Fotemustine proved to be significantly more active than BCNU in IGRM34 and the glioma xenograft IGRG88. The DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) was detected in all tumor xenografts, ranging in activity from 6 to 892 fmol/mg protein. The high in vivo sensitivity to fotemustine and BCNU observed in three xenografts was clearly associated with a low ATase activity (> 20 fmol/mg), whereas the two poorly sensitive or refractory medulloblastoma xenografts showed high ATase activity (> 500 fmol/mg). Alkylpurine-DNA N-glycosylase activity was detected in all tumor xenografts but at levels ranging only from 513 to 1105 fmol/mg/h; no consistent relationship was found between alkylpurine-DNA N-glycosylase activity and the in vivo sensitivity to the two chloroethylnitrosoureas. The improved activity and tolerance of fotemustine in comparison with BCNU in pediatric medulloblastoma xenografts strongly support the clinical development of this agent in children with brain tumors, in which ATase should be examined as a potential prognostic indicator.

The purpose of this study was to find out whether the glutathione (GSH), in red blood cells could predict the response to neoadjuvant chemotherapy cisplatin/5-fluorouracil (CDDP/5-FU) in patients with head and neck squamous cell carcinoma (HNSCC). Three courses of induction chemotherapy with CDDP/5-FU were administered and followed by surgery and radiotherapy or radiotherapy alone, in 51 patients with HNSCC. GSH was measured by spectrophotometry in red blood cell before any treatment (Sample 1: S1), after each course of chemotherapy (S2, S3, S4). Our results showed that GSH was the same at diagnosis in patients with complete or partial response (OR) compared to those with stable or progressive disease (NR). With regard to evolution of the GSH during the 3 courses of CT a significant difference was found between courses (S2: 5.06 +/- 0.35 vs S4 = 3.61 +/- 0.4 mumol/g haemoglobin, p < 0.05). When we separated our patients into OR and NR, a significant difference was found over the 3 courses of chemotherapy for GSH content. Non responder patients showed decreased GSH content at the end of the treatment, (S2: 5 +/- 0.5 vs S4: 2.2 +/- 0.4 mumol/g haemoglobin, p < 0.05) while OR were stable. In conclusion, red blood cell GSH seems to have no early predictive value for chemoresponse to neoadjuvant chemotherapy CDDP/5-FU in HNSCC.

Hypercholesterolemia increases the oxidation of low density lipoprotein (LDL) which subsequently leads to atherogenesis. The oxidized LDL are also known to increase in vitro macrophage synthesis of glutathione. The purpose of this study was to investigate the relationship between lipid parameters and the glutathione system (glutathione, glutathione S-transferase) in total blood and within leukocytes. The glutathione and glutathione S-transferase were evaluated by spectrophotometric methods in sixty-two healthy volunteers (32 women, 30 men, mean age 39.9 +/- 7.7). No correlation was found between the level of blood cholesterol and the values of the blood glutathione system. However, a positive correlation between the values of glutathione and glutathione S-transferase in leukocytes and the blood cholesterol level was only found in women (r = 0.55 and r = 0.50 respectively, p < 0.01). We also found in men a positive correlation between body mass index and glutathione S-transferase in total blood and within leukocytes (r = 0.38, p < 0.05, r = 0.5, p < 0.01 respectively). No correlation was found between age, smoking and the values of the glutathione system. Our results suggest that the glutathione system in leukocytes is related to blood cholesterol levels. The fact that this positive correlation was only observed in women points to a possible role of estrogens in the regulation of the glutathione system which merits to be further studied.

BACKGROUND:Scleroderma patients exhibit increased chromosomal instability due to circulating clastogenic plasma factors (CF). Formation and action mechanisms of CF are mediated by superoxide. In addition, previous work detected inosine triphosphate (ITP) in the plasma of 2 patients, and the enzyme adenosine deaminase (ADA) was found to be increased.OBJECTIVE:To study correlations between CF, ITP and ADA levels, CF and disease activity, as well as other biomarkers of oxidative stress.METHODS:Clastogenic activity was evaluated by means of cytogenetic methods in 48 patients and 55 healthy subjects. ITP was detected by mass spectrometry and electrospray ionisation. ADA was measured with a colorimetric assay and malondialdehyde using the Yagi method.RESULTS:Clastogenic activity was significantly increased in patients' plasma compared to controls. In 10 patients CF, ITP and ADA were studied simultaneously. All three parameters were increased in the 7 patients of subgroups 2 (skin and esophagus involvement) and 3 (skin plus multiple organ involvement). ITP was not detected in 2 patients of subgroup 1 (skin involvement only) with low ADA and CF values.CONCLUSION:ITP, the deamination product of ATP, is one of the clastogenic and superoxide generating components of CF. The formation of this deamination product of ATP is probably related to the increase in ADA. CF are biomarkers of oxidative stress and can be used for evaluation of antioxidant treatments in scleroderma.

Pharmacogenetics could be defined as the study of genetically controlled variations in drug response. Introduction of pharmacogenetics in hematology and oncology has been done recently. With recombinant DNA technology, like restriction analysis of genomic DNA, enzymatic amplification of DNA by the polymerase chain reaction and expression of cDNAs in cell cultures, this research area has been developed during the last 10 years. In hematology and oncology, we can integrate pharmacogenetics in 3 areas. First, the concept of genetic risk of cancer and the study of drug or carcinogen metabolizing enzymes that could modulate this risk, regarding the activity of some specific enzymes; second, the use of pharmacogenetics, related to the toxicity or efficacy of anticancer drugs, allowing the identification of key enzymes involved in the biotransformation of the drug and the study of molecular aspects involved in the regulation of the activity of the enzymes; third, the implication of the study of enzymatic activities in tumoral tissues as compared to non-tumoral tissues. The following differences between the 2 tissues can be subsequently used to increase the specificity of the anticancer drugs.