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1.
J Nanobiotechnology ; 19(1): 74, 2021 Mar 16.
Article in English | MEDLINE | ID: mdl-33726759

ABSTRACT

BACKGROUND: This research was to develop a special method for enriching Circulating tumor cells (CTCs) of Hepatocellular carcinoma (HCC) by Glypican-3 immunoliposomes (GPC3-IML), and to analyze the correlation between the CTCs count and tumor malignancy, as well as to investigate the mutation characteristics of CTC-derived NGS. RESULTS: In this study characterization of physical parameters was performed with the preparation of GPC3-IML. CTCs in peripheral blood of HCC patients were further separated and identified. Immunofluorescence was used to identify CTCs for further counting. By this means, the correlation between CTCs count and clinicopathological features was analyzed, and the genetic mutation characteristics of NGS derived from CTCs were investigated and compared with that of tissue NGS. Results showed that compared with EpCAM and vimentin, GPC-3 had a stronger CTCs separation ability. There was a correlation between "positive" count of CTCs (≥ 5 PV-CTC per 7.5 ml blood) and BCLC stage (P = 0.055). The result of CTC-NGS was consistent with that of tissue-NGS in 60% cases, revealing that KMT2C was a common highly-frequent mutated gene. CONCLUSION: The combination of immunomagnetic separation of CTCs and anti-tumor marker identification technology can be regarded as a new technology of CTCs detection in peripheral blood of patients with HCC. Trial registration EHBHKY2020-k-024. Registered 17 August 2020-Retrospectively registered.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/metabolism , Carcinoma, Hepatocellular/metabolism , Glypicans/metabolism , Liver Neoplasms/metabolism , Adult , Aged , Biomarkers, Tumor/blood , Carboplatin/metabolism , Carcinoma, Hepatocellular/pathology , Cyclophosphamide/metabolism , Epithelial Cell Adhesion Molecule , Female , High-Throughput Nucleotide Sequencing , Humans , Liver Neoplasms/pathology , Male , Middle Aged , Neoplastic Cells, Circulating , Thiotepa/metabolism , Young Adult
2.
Int J Clin Pharmacol Ther ; 53(11): 914-22, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26396136

ABSTRACT

BACKGROUND: The shifts to second-line chemotherapy for metastatic breast cancer (MBC) were widely required based on pharmaceutical molecular profiles to reach out precision medicine. The emerging precise treatment of cancer requires the implementation of clarified pharmacogenetic profiles which are capable of elucidating the predictive responses to cancer chemotherapy. Therefore we were interested in the analysis of the roles of single nucleotide polymorphism (SNP) of GSTP1 (glutathione S-transferase pi 1 gene) alleles to identify pharmacological links with predictors of clinical responses and toxicities. METHODS: 93 MBC patients receiving thiotepa plus docetaxel chemotherapy were enrolled in this study. Optimized CYP3A5, CYP2B6, and GSTP1 were predominantly selected as candidate genes and their three SNPs (CYP2B6 G516T, CYP3A5 A6986G, and GSTP1 A313G) were genotyped by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry. Progression-free survival (PFS), disease control rate, and chemo-related toxicities were recorded. RESULTS: GSTP1 A313G (rs1695) was identified to be related with disease progression. In particular, patients harboring AG/GG genotype demonstrated a statistically longer PFS than those with AA. Multivariate analysis confirmed that AG/GG genotype was associated with both clinical responses and liver-localized metastatic lesions. No correlation was found between these three SNPs and chemotherapy-induced toxicity. CONCLUSIONS: These results suggest that the GSTP1 polymorphism is a novel prognostic marker for clinical response to thiotepa-containing chemotherapy regimens. Such evidence could provide insight into the role of pharmacogenetics to deprive of biases in shifting regimens solely by empirical choices.


Subject(s)
Antineoplastic Agents, Alkylating/therapeutic use , Breast Neoplasms/drug therapy , Glutathione S-Transferase pi/genetics , Polymorphism, Single Nucleotide , Thiotepa/therapeutic use , Triethylenephosphoramide/therapeutic use , Aged , Antineoplastic Agents, Alkylating/adverse effects , Antineoplastic Agents, Alkylating/metabolism , Biotransformation , Breast Neoplasms/pathology , Chi-Square Distribution , China , Cytochrome P-450 CYP2B6/genetics , Cytochrome P-450 CYP2B6/metabolism , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A/metabolism , Disease Progression , Disease-Free Survival , Female , Genotype , Glutathione S-Transferase pi/metabolism , Humans , Logistic Models , Middle Aged , Multivariate Analysis , Neoplasm Metastasis , Odds Ratio , Patient Selection , Pharmacogenetics , Phenotype , Retrospective Studies , Risk Factors , Salvage Therapy , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Thiotepa/adverse effects , Thiotepa/metabolism , Time Factors , Treatment Outcome , Triethylenephosphoramide/adverse effects , Triethylenephosphoramide/metabolism
3.
Acta Trop ; 132 Suppl: S164-9, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24513037

ABSTRACT

Successful SIT trials against mosquitoes in the 1960-70s were achieved by sterilizing male mosquitoes using chemosterilants. Their use was discontinued after concerns were raised about the effect of residues on non-target organisms, although scant evidence has been published. Irradiation is an expensive process; chemosterilization could be an affordable option for implementing SIT programs in developing countries. We compare life table parameters of three Aedes aegypti populations comprising different ratios of thiotepa-treated and non-treated males in order to identify the impact on reproductive potential of the presence of sterile males. No difference was observed in the survival of the treated and untreated males. The release of thiotepa sterilized males into caged Ae. aegypti populations had no effect on death or survival probability of the individuals in the cages but the fecundity of females was significantly reduced, as evaluated by hatch rate and stable age structure parameters. The significant decreases in net reproduction rate, finite rate of natural increase and intrinsic rate of natural increase in populations including sterile males are sufficient to indicate that such populations would not be able to proliferate in natural conditions. This suggests that release of Ae. aegypti thiotepa-treated males could be effective in reducing the reproductive capability of the target population and consequently contribute to vector control.


Subject(s)
Aedes/drug effects , Aedes/growth & development , Chemosterilants/metabolism , Sterilization, Reproductive/methods , Thiotepa/metabolism , Animals , Female , Fertility , Life Tables , Male , Pest Control, Biological/methods , Reproduction
4.
Biochem Pharmacol ; 81(8): 1043-53, 2011 Apr 15.
Article in English | MEDLINE | ID: mdl-21300029

ABSTRACT

ThioTEPA, an alkylating agent with anti-tumor activity, has been used as an effective anticancer drug since the 1950s. However, a complete understanding of how its alkylating activity relates to clinical efficacy has not been achieved, the total urinary excretion of thioTEPA and its metabolites is not resolved, and the mechanism of formation of the potentially toxic metabolites S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA) remains unclear. In this study, the metabolism of thioTEPA in a mouse model was comprehensively investigated using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based-metabolomics. The nine metabolites identified in mouse urine suggest that thioTEPA underwent ring-opening, N-dechloroethylation, and conjugation reactions in vivo. SCMC and TDGA, two downstream thioTEPA metabolites, were produced from thioTEPA from two novel metabolites 1,2,3-trichloroTEPA (VII) and dechloroethyltrichloroTEPA (VIII). SCMC and TDGA excretion were increased about 4-fold and 2-fold, respectively, in urine following the thioTEPA treatment. The main mouse metabolites of thioTEPA in vivo were TEPA (II), monochloroTEPA (III) and thioTEPA-mercapturate (IV). In addition, five thioTEPA metabolites were detected in serum and all shared similar disposition. Although thioTEPA has a unique chemical structure which is not maintained in the majority of its metabolites, metabolomic analysis of its biotransformation greatly contributed to the investigation of thioTEPA metabolism in vivo, and provides useful information to understand comprehensively the pharmacological activity and potential toxicity of thioTEPA in the clinic.


Subject(s)
Antineoplastic Agents, Alkylating/metabolism , Carbocysteine/metabolism , Metabolomics/methods , Thioglycolates/metabolism , Thiotepa/metabolism , Animals , Antineoplastic Agents, Alkylating/blood , Antineoplastic Agents, Alkylating/urine , Carbocysteine/blood , Carbocysteine/urine , Chromatography, Liquid , Male , Metabolomics/instrumentation , Mice , Mice, Inbred C57BL , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Multivariate Analysis , Spectrometry, Mass, Electrospray Ionization , Thioglycolates/blood , Thioglycolates/urine , Thiotepa/blood , Thiotepa/urine
5.
Front Biosci (Elite Ed) ; 3(1): 240-9, 2011 01 01.
Article in English | MEDLINE | ID: mdl-21196304

ABSTRACT

It has been suggested that breast cancer stem cells (CSCs), which characterized by CD44(+)CD24(-/low), may result in treatment failure in patients with breast cancer. It is possible therefore that that inhibiting such subpopulation might subsequently improve clinical outcome. In the present study, we found that the CD44(+)/CD24(-/low) CSCs, isolated from both human breast cell line MCF-7 and MDA-MB-231, were more resistant to thiotepa, paclitaxel and anthracycline, when compared with the non-breast cancer stem cell subset from the same cell lines, whereas the chemosensitivities were remarkably reversed by higher concentration of thiotepa and paclitaxel except for adriamycin. The percentage of CSCs was significantly decreased with an addition of DNA methyltransferase inhibitor CDA-2 and the expression of Smo, Shh, and Gli-1 of Hedgehog signaling pathway in CSCs was decreased. Of important findings, combination of thiotepa or paclitaxel with CDA-2 could significantly inhibit the proliferation of CSCs regardless of their dosages. These results unveiled that the selection of cytotoxic agents and increasing their dosage might be of great importance in the respect of eliminating CSCs. DNA methyltransferase inhibitor CDA-2 exhibited a synergistic effect with cytotoxic drugs, which might provide a conceptually new therapeutic strategy.


Subject(s)
Breast Neoplasms/drug therapy , DNA Modification Methylases/antagonists & inhibitors , Neoplastic Stem Cells/drug effects , Paclitaxel/pharmacology , Peptides/pharmacology , Phenylacetates/pharmacology , Thiotepa/pharmacology , CD24 Antigen/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Primers/genetics , Dose-Response Relationship, Drug , Drug Synergism , Female , Flow Cytometry , Hedgehog Proteins/metabolism , Humans , Hyaluronan Receptors/metabolism , Paclitaxel/metabolism , Peptides/metabolism , Phenylacetates/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Thiotepa/metabolism
6.
J Mol Model ; 16(8): 1383-90, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20155478

ABSTRACT

Density functional theory (DFT) using the B3LYP functional was applied to elucidate the molecular properties of the antitumor drug thiotepa and its main metabolite tepa. Aqueous solvent effects were introduced using the conductor-like polarizable continuum model (CPCM). The protocol for calculating the pK (a) values obtained with different cavity models was tested on a series of aziridine and phosphoramide compounds. An efficient computational scheme has been identified that uses the CPCM model of solvation with a universal force field (UFF) cavity. The method has been used to evaluate the basicities of thiotepa and its metabolite. Our calculations show that the basicities of the aziridine moiety of thiotepa and tepa are dramatically reduced compared to free aziridine, indicating that highly acidic media are needed to produce substantial yields of the N-protonated form of the drug. Finally, the mechanisms of reaction of the drug and its metabolite are discussed based on our theoretical results. The calculations reproduce the experimental trends very satisfactorily.


Subject(s)
Antineoplastic Agents/chemistry , Chemical Phenomena , Models, Chemical , Thiotepa/analogs & derivatives , Thiotepa/chemistry , Antineoplastic Agents/metabolism , Aziridines/chemistry , DNA/metabolism , Electrons , Gases/chemistry , Models, Molecular , Protons , Solvents , Static Electricity , Thermodynamics , Thiotepa/metabolism
7.
Cancer Chemother Pharmacol ; 56(4): 370-8, 2005 Oct.
Article in English | MEDLINE | ID: mdl-15838656

ABSTRACT

BACKGROUND: Patients receiving the highly emetogenic high-dose chemotherapy regimen with cyclophosphamide, thiotepa and carboplatin (CTC) may benefit from the neurokin-1 receptor antagonist aprepitant in addition to standard anti-emetic therapy. As aprepitant has been shown to be a moderate inhibitor of the cytochrome P450 (CYP) 3A4 isoenzyme, its effect on the pharmacokinetics and metabolism of cyclophosphamide and thiotepa was evaluated. Moreover, preliminary results on the clinical efficacy of aprepitant in the CTC regimen are reported. PATIENTS AND METHODS: Six patients were enrolled in a protocol that employed a 4-day course of CTC high-dose chemotherapy with cyclophosphamide (1,500 mg/m2/day), thiotepa (120 mg/m2/day) and carboplatin (AUC 5 mg min/ml/day). Two patients received the tCTC protocol, which comprises two-third of the dose of CTC. In addition to standard anti-emetic therapy, the patients received aprepitant from one day before the start of their course until 3 days after chemotherapy. Blood samples were collected on days one and three of the course and analyzed for cyclophosphamide and its activated metabolite 4-hydroxycyclophosphamide, thiotepa and its main active metabolite tepa. The influence of aprepitant on the pharmacokinetics of cyclophosphamide and thiotepa was analyzed using a population pharmacokinetic analysis including a reference population of 49 patients receiving the same chemotherapy regimen without aprepitant and sampled under the same conditions. The frequency of nausea and vomiting in the six patients receiving CTC was compared with those of the last 22 consecutive patients receiving CTC chemotherapy without aprepitant. Inhibitory activity of aprepitant on cyclophosphamide and thiotepa metabolism was also tested in human liver microsomes. RESULTS: In our patient population, the rate of autoinduction of cyclophosphamide (P=0.040) and the formation clearance of tepa (P<0.001) were reduced with 23% and 33% when aprepitant was co-administered, respectively. Exposures to the active metabolite 4-hydroxycyclophosphamide and tepa were therefore reduced (5% and 20%, respectively) in the presence of aprepitant. In human liver microsomes, the 50% inhibitory concentrations (IC50) of aprepitant for inhibition of cyclophosphamide (IC50=1.3 microg/ml) and thiotepa (IC50=0.27 microg/ml) metabolism were within the therapeutic range. Patients receiving aprepitant experienced less frequently CINV both during and after the CTC course compared with the reference population (nausea 3.7 days vs. 5.8 days, P=0.052; vomiting 0.5 days vs. 4.8 days, P<0.001). CONCLUSION: Aprepitant inhibited both cyclophosphamide and thiotepa metabolism, most probably due to inhibition of the CYP 3A4 and/or 2B6 isoenzymes. The effects of this interaction are, however, small compared to the total variability. Addition of aprepitant may provide superior protection against vomiting in patients receiving the highly emetogenic high-dose CTC chemotherapy.


Subject(s)
Antiemetics/pharmacology , Breast Neoplasms/metabolism , Carboplatin/pharmacokinetics , Cyclophosphamide/pharmacokinetics , Cytochrome P-450 Enzyme Inhibitors , Morpholines/pharmacology , Neoplasms, Germ Cell and Embryonal/metabolism , Thiotepa/metabolism , Antiemetics/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Aprepitant , Breast Neoplasms/drug therapy , Carboplatin/administration & dosage , Carboplatin/metabolism , Cyclophosphamide/administration & dosage , Cyclophosphamide/metabolism , Drug Interactions , Female , Humans , Male , Microsomes, Liver/metabolism , Morpholines/therapeutic use , Nausea/chemically induced , Nausea/prevention & control , Neoplasms, Germ Cell and Embryonal/drug therapy , Thiotepa/administration & dosage , Thiotepa/pharmacokinetics
8.
Cancer Chemother Pharmacol ; 55(5): 507-10, 2005 May.
Article in English | MEDLINE | ID: mdl-15685452

ABSTRACT

PATIENT AND METHOD: A 42-year-old male patient with relapsing germ-cell cancer was enrolled in a salvage protocol that employed two 4-day courses of CTC high-dose chemotherapy with cyclophosphamide (1,500 mg m(-2) day(-1)), thiotepa (120 mg m(-2) day(-1)), and carboplatin, followed by peripheral blood progenitor cell support. From five days before the start of the second CTC course the patient received phenytoin for generalized epileptic seizures. Blood samples were collected on day 1 of both CTC courses and analyzed for cyclophosphamide and its activated metabolite 4-hydroxycyclophosphamide, and for thiotepa and its main active metabolite tepa. RESULTS: Exposure (expressed as area under the plasma concentration vs time curve) to 4-hydroxycyclophosphamide and tepa in the second CTC course was increased by 51% and 115%, respectively, compared with the first CTC course, whereas exposure to cyclophosphamide and thiotepa was significantly reduced (67% and 29%, respectively). Because high exposure to 4-hydroxycyclophosphamide and tepa correlates with increased toxicity, the treatment risk of this patient was significantly increased. Therefore doses were reduced on the third day of the second course. CONCLUSION: It was concluded that phenytoin significantly induces both cyclophosphamide and thiotepa metabolism, most probably by induction of the cytochrome p450 enzyme system. This potential clinical significant interaction should be taken into account when phenytoin is administered in combination with cyclophosphamide and thiotepa in clinical practice.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/metabolism , Cyclophosphamide/metabolism , Neoplasms, Germ Cell and Embryonal/metabolism , Phenytoin/pharmacology , Thiotepa/metabolism , Adult , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carboplatin/administration & dosage , Cyclophosphamide/therapeutic use , Cytochrome P-450 Enzyme System/biosynthesis , Cytochrome P-450 Enzyme System/physiology , Drug Interactions , Enzyme Induction/drug effects , Humans , Male , Neoplasms, Germ Cell and Embryonal/drug therapy , Salvage Therapy , Thiotepa/therapeutic use
9.
Drug Metab Dispos ; 32(9): 930-6, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15319333

ABSTRACT

Meperidine is an opioid analgesic metabolized in the liver by N-demethylation to normeperidine, a potent stimulant of the central nervous system. The purpose of this study was to identify the human cytochrome P450 (P450) enzymes involved in normeperidine formation. Our in vitro studies included 1) screening 16 expressed P450s for normeperidine formation, 2) kinetic experiments on human liver microsomes and candidate P450s, and 3) correlation and inhibition experiments using human hepatic microsomes. After normalization by its relative abundance in human liver microsomes, CYP2B6, CYP3A4, and CYP2C19 accounted for 57, 28, and 15% of the total intrinsic clearance of meperidine. CYP3A5 and CYP2D6 contributed to < 1%. Formation of normeperidine significantly correlated with CYP2B6-selective S-mephenytoin N-demethylation (r = 0.88, p < 0.0001 at 75 > microM meperidine, and r = 0.89, p < 0.0001 at 350 microM meperidine, n = 21) and CYP3A4-selective midazolam 1'-hydroxylation (r = 0.59, p < 0.01 at 75 microM meperidine, and r = 0.55, p < 0.01 at 350 microM meperidine, n = 23). No significant correlation was observed with CYP2C19-selective S-mephenytoin 4'-hydroxylation (r = 0.36, p = 0.2 at 75 microM meperidine, and r = 0.02, p = 0.9 at 350 microM meperidine, n = 13). An anti-CYP2B6 antibody inhibited normeperidine formation by 46%. In contrast, antibodies inhibitory to CYP3A4 and CYP2C8/9/18/19 had little effect (<14% inhibition). Experiments with thiotepa and ketoconazole suggested inhibition of microsomal CYP2B6 and CYP3A4 activity, whereas studies with fluvoxamine (a substrate of CYP2C19) were inconclusive due to lack of specificity. We conclude that normeperidine formation in human liver microsomes is mainly catalyzed by CYP2B6 and CYP3A4, with a minor contribution from CYP2C19.


Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 Enzyme System/metabolism , Meperidine/analogs & derivatives , Meperidine/metabolism , Microsomes, Liver/enzymology , Mixed Function Oxygenases/metabolism , Oxidoreductases, N-Demethylating/metabolism , Aryl Hydrocarbon Hydroxylases/genetics , Cytochrome P-450 CYP2B6 , Cytochrome P-450 CYP2C19 , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , Fluvoxamine/metabolism , Fluvoxamine/pharmacology , Humans , Hydroxylation/drug effects , Isoenzymes/metabolism , Ketoconazole/metabolism , Ketoconazole/pharmacology , Meperidine/pharmacology , Mephenytoin/chemistry , Mephenytoin/metabolism , Mixed Function Oxygenases/genetics , Oxidoreductases, N-Demethylating/genetics , Recombinant Proteins/metabolism , Thiotepa/metabolism , Thiotepa/pharmacology
10.
J Pharmacol Exp Ther ; 310(3): 1011-9, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15121764

ABSTRACT

The anticancer drug N,N,"N"-triethylenethiophosphoramide (tTEPA) inactivated CYP2B6 and CYP2B1 in the reconstituted system in a time-, concentration-, and NADPH-dependent manner indicative of mechanism-based inactivation. The KI value for the inactivation of CYP2B1 was 38 microM, the kinact was 0.3 min(-1), and the t1/2 value was 2.5 min. Spectral carbon monoxide (CO) binding and high-performance liquid chromatography heme studies of the tTEPA-inactivated CYP2B1 suggest that the loss in the enzymatic activity was primarily due to the binding of a reactive tTEPA intermediate to the 2B1 apoprotein. Inactivation by tTEPA in the presence of 7-ethoxycoumarin, an alternate substrate, reduced the rate of inactivation of CYP2B1. Incubations with tTEPA and NADPH resulted in greater than 90% loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation and testosterone hydroxylation activity of CYP2B1. In contrast, benzphetamine metabolism was significantly less inhibited (47%). CYP2B6 was inactivated by tTEPA with a KI value of 50 microM, a k inact value of 0.1 min(-1), and a t1/2 value of 14 min. However, unlike CYP2B1, the tTEPA-inactivated human isoform showed losses in the cytochrome P450 (P450) CO spectrum, the pyridine hemochrome spectrum, and in the amount of native heme that were comparable with the loss in the 7-EFC and benzphetamine activity, suggesting that activity loss was brought about by a tTEPA-reactive intermediate damaging the CYP2B6 heme. CYP2B6 could only be protected from the tTEPA-dependent inactivation by the 2B6-specific substrate bupropion but not by other substrates of CYP2B such as benzphetamine, testosterone, or 7-ethoxycoumarin. The data indicate that tTEPA metabolism by these two 2B isoforms results in inactivation of the P450s by two distinct mechanisms.


Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 CYP2B1/metabolism , Enzyme Inhibitors/metabolism , Isoenzymes/metabolism , Oxidoreductases, N-Demethylating/metabolism , Thiotepa/metabolism , Amines/metabolism , Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Benzphetamine/metabolism , Cytochrome P-450 CYP2B1/antagonists & inhibitors , Cytochrome P-450 CYP2B6 , Enzyme Inhibitors/pharmacology , Humans , Isoenzymes/antagonists & inhibitors , Oxidoreductases, N-Demethylating/antagonists & inhibitors , Testosterone/metabolism , Thiotepa/pharmacology
11.
Bone Marrow Transplant ; 30(9): 593-7, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12407434

ABSTRACT

Reversible alopecia is a commonly observed, important and distressing complication of chemotherapy. Permanent alopecia, however, is rare after standard-dose therapy, but has occasionally been observed after high-dose chemotherapy with cyclophosphamide, thiotepa and carboplatin (CTC). We evaluated the relationships between total exposure to these three compounds and their different metabolites in the high-dose CTC regimen, and the subsequent development of irreversible alopecia. Twenty-four patients received two or three courses of high-dose CTC, each followed by peripheral blood progenitor cell transplantation. Plasma levels of cyclophosphamide, its active metabolite 4-hydroxycyclophosphamide, thiotepa, its active metabolite tepa, and carboplatin were determined, and the area-under-the-plasma concentration-versus-time curves (AUC) of the compounds were calculated. Eight of the 24 patients included in the study developed permanent alopecia, while seven had normal hair regrowth and nine patients developed incomplete and/or thin hair regrowth. The carboplatin AUC and the summed AUC of thiotepa and tepa were both significantly associated with increasing irreversibility of hair loss. These results suggest that high exposure to carboplatin and the sum of the thiotepa and tepa exposure may lead to the development of permanent alopecia. This knowledge could guide therapeutic drug monitoring in order to prevent the occurrence of permanent alopecia and thereby improve the patients' quality of life.


Subject(s)
Alopecia/chemically induced , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Cyclophosphamide/analogs & derivatives , Adolescent , Adult , Alopecia/etiology , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Area Under Curve , Carboplatin/pharmacokinetics , Carboplatin/pharmacology , Cyclophosphamide/blood , Cyclophosphamide/metabolism , Cyclophosphamide/pharmacokinetics , Cyclophosphamide/pharmacology , Drug Evaluation , Female , Humans , Male , Middle Aged , Neoplasms/complications , Neoplasms/drug therapy , Thiotepa/metabolism , Thiotepa/pharmacokinetics , Thiotepa/pharmacology , Triethylenephosphoramide/blood
12.
Cancer Chemother Pharmacol ; 49(6): 461-7, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12107550

ABSTRACT

PURPOSE: To establish the cytochrome P450 (CYP) isozymes involved in the metabolism of the alkylating agent, thiotepa, to the pharmacologically active metabolite, TEPA. METHODS: In vitro chemical inhibition studies were conducted by incubating thiotepa and pooled human hepatic microsomes in the presence of known inhibitors to CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Studies were also performed with cloned, expressed CYP3A4, CYP2A6, CYP2E1 and CYP2B6 microsomes, and anti-CYP2B6 monoclonal antibody. RESULTS: Known CYP3A4 inhibitors reduced TEPA production. Inhibition with CYP2E1 inhibitors was inconsistent. All other inhibitors produced little or no change in TEPA formation. Cloned, expressed CYP2B6 and CYP3A4 microsomes catalyzed TEPA formation, whereas CYP2A6 and CYP2E1 did not. Incubation of thiotepa with anti-CYP2B6 antibody and cloned, expressed CYP2B6 microsomes resulted in reductions in the formation of TEPA, but no change in TEPA formation occurred in human liver microsomes. CONCLUSIONS: Thiotepa is metabolized in human liver microsomes by CYP3A4 (major) and CYP2B6 (minor). There is a potential for CYP-mediated drug interactions with thiotepa. Pharmacokinetic variability of thiotepa may be related to expression of hepatic CYP isozymes.


Subject(s)
Antineoplastic Agents, Alkylating/metabolism , Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/metabolism , Microsomes, Liver/enzymology , Mixed Function Oxygenases/metabolism , Oxidoreductases, N-Demethylating/metabolism , Thiotepa/metabolism , Triethylenephosphoramide/metabolism , Antibodies/pharmacology , Cytochrome P-450 CYP2B6 , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/pharmacology , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Kinetics , Microsomes, Liver/drug effects , Mixed Function Oxygenases/antagonists & inhibitors , Oxidoreductases, N-Demethylating/antagonists & inhibitors , Thiotepa/antagonists & inhibitors , Transfection
13.
Anticancer Drugs ; 12(6): 519-24, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11459998

ABSTRACT

The urinary excretion of N,N',N"-triethylenethiophosphoramide (thioTEPA), and its metabolites N,N',N"-triethylenephosphoramide (TEPA), N,N'-diethylene,N"-2-chloroethylphosphoramide (monochloroTEPA) and thioTEPA--mercapturate was determined in patients receiving thioTEPA as part of a high-dose combination chemotherapy regimen with cyclophosphamide and carboplatin. The thioTEPA dose was 40 or 60 mg/m(2) in short infusions, twice daily, during 4 days. Urine samples were collected after each voiding on each day of drug administration until 24--48 h after the last thioTEPA infusion. ThioTEPA, TEPA and monochloroTEPA concentrations were determined with gas chromatography and thioTEPA--mercapturate with liquid chromatography--mass spectrometry with direct sample injection. ThioTEPA was present in urine 30 min after infusion and was still excreted 18 h after the last infusion. All metabolites were detected in urine 1 h after infusion. Patients with a creatinine clearance above 140 ml/minl showed higher excretion of TEPA than patients with a creatinine clearance below 140 ml/min (12.8 versus 4.9%, p=0.01). The excretion of monochloroTEPA relative to the excreted amount of TEPA increased at lower pH values of the urine. The excretion of thioTEPA--mercapturate relative to the dose was higher in patients treated with 60 mg/m(2). Excretion of thioTEPA and monochloroTEPA both accounted for only 0.5% of the dose, while TEPA and thioTEPA--mercapturate both accounted for 11.1%.


Subject(s)
Acetylcysteine/analogs & derivatives , Acetylcysteine/urine , Antineoplastic Agents/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Aziridines/urine , Carboplatin/administration & dosage , Cyclophosphamide/administration & dosage , Thiotepa/administration & dosage , Thiotepa/metabolism , Thiotepa/urine , Adolescent , Adult , Antineoplastic Agents, Alkylating/administration & dosage , Breast Neoplasms/urine , Creatinine/urine , Dose-Response Relationship, Drug , Female , Humans , Hydrogen-Ion Concentration , Male , Middle Aged , Models, Chemical , Neoplasms, Germ Cell and Embryonal/urine , Time Factors , Triethylenephosphoramide/urine
14.
Anticancer Res ; 20(3A): 1711-6, 2000.
Article in English | MEDLINE | ID: mdl-10928097

ABSTRACT

BACKGROUND: The combination of cyclophosphamide, thioTEPA and carboplatin is used in our Institute for the treatment of breast or germ cell cancer. ThioTEPA inhibits the bioactivation of cyclophosphamide, and platinum drugs are known to interfere with the hepatic metabolism of several anticancer drugs. Of the co-administered drugs to prevent unwanted side effects, some are enzyme inducers, cytochrome P450 inhibitors or substrates. The aim of this study was to investigate the influence of co-medicated drugs on the biotransformation of thioTEPA. METHODS: The possible inhibition of the metabolism of thioTEPA to TEPA was investigated in human microsomes. Influences on the conversion of thioTEPA to monoglutathionylthioTEPA, was studied by the incubation of thioTEPA with glutathione and glutathione S-transferase. RESULTS: No inhibition of the metabolism of thioTEPA to form TEPA was observed for cyclophosphamide and carboplatin, or any other co-medicated drug (ciproflocaxin, amphotericin B, itraconazol, fluconazol, ondansetron, dexamethasone, granisetron, aciclovir, ranitidine, lorazepam). The conversion of thioTEPA to monoglutathionylthioTEPA was inhibited by cyclophosphamide, itraconazol, amphotericin B and ondansetron with IC50 values of 58, 256, 55 and 40 mM, respectively, which are far higher than therapeutic drug levels. CONCLUSION: No clinically relevant drug-drug interactions occur in the CTC regimen as applied in our Institute.


Subject(s)
Acetylcysteine/metabolism , Carboplatin/pharmacology , Microsomes/drug effects , Thiotepa/metabolism , Triethylenephosphoramide/metabolism , Antineoplastic Agents/pharmacology , Biotransformation/drug effects , Cyclophosphamide/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Drug Interactions , Glutathione/metabolism , Humans , In Vitro Techniques , Kinetics , Microsomes/metabolism
15.
Int J Pharm ; 200(2): 187-94, 2000 May 10.
Article in English | MEDLINE | ID: mdl-10867248

ABSTRACT

The degradation of N,N',N"-triethylenethiophosphoramide (thioTEPA) and its metabolites N,N',N"-triethylenephosphoramide (TEPA), N, N'-diethylene,N"-2-chloroethylphosphoramide (monochloroTEPA) and thioTEPA-mercapturate in plasma and urine has been investigated. ThioTEPA, TEPA and monochloroTEPA were analyzed using a gas chromatographic (GC) system with selective nitrogen/phosphorous detection; thioTEPA-mercapturate was analyzed on a liquid chromatography-mass spectrometric (LC-MS) system. The influences of pH and temperature on the stability of thioTEPA and its metabolites were studied. An increase in degradation rate was observed with decreasing pH as measured for all studied metabolites. In urine the rate of degradation at 37 degrees C was approximately 2.5+/-1 times higher than at 22 degrees C. At 37 degrees C thioTEPA and TEPA were more stable in plasma than in urine, with half lives ranging from 9-20 h for urine and 13-34 h for plasma at pH 6. Mono- and dichloro derivatives of thioTEPA were formed in urine and the monochloro derivative was found in plasma. Degradation of TEPA in plasma and urine resulted in the formation of monochloroTEPA. During the degradation of TEPA in plasma also the methoxy derivative of TEPA was formed as a consequence of the applied procedure. The monochloro derivative of thioTEPA-mercapturate was formed in urine, whereas for monochloroTEPA no degradation products could be detected.


Subject(s)
Acetylcysteine/analogs & derivatives , Aziridines/metabolism , Thiotepa/metabolism , Triethylenephosphoramide/metabolism , Acetylcysteine/blood , Acetylcysteine/metabolism , Acetylcysteine/urine , Antineoplastic Agents, Alkylating/blood , Antineoplastic Agents, Alkylating/chemistry , Antineoplastic Agents, Alkylating/metabolism , Antineoplastic Agents, Alkylating/urine , Aziridines/blood , Aziridines/urine , Drug Stability , Mass Spectrometry , Thiotepa/blood , Thiotepa/chemistry , Thiotepa/urine , Triethylenephosphoramide/blood , Triethylenephosphoramide/chemistry , Triethylenephosphoramide/urine
16.
J Chromatogr B Biomed Sci Appl ; 732(1): 73-9, 1999 Sep 10.
Article in English | MEDLINE | ID: mdl-10517224

ABSTRACT

An assay for the quantitative determination of the mercapturic acid conjugate of N,N',N"-triethylenethiophosphoramide (thioTEPA-mercapturate) in human urine has been developed. ThioTEPA-mercapturate, a recently identified metabolite of the alkylating anticancer agent thioTEPA, was analyzed using LC-MS and with direct sample injection. Sulphadiazine was used as internal standard. Linearity was accomplished in the therapeutic relevant range of 1-25 microg/ml; recovery was 84% and both accuracy and precision were less than 20% for the lower limit of quantification (1.0 microg/ml) and less than 10% for the other concentration levels. The stability of thioTEPA-mercapturate proved to be satisfactory over a period of 2 months, when kept at -80 degrees C. ThioTEPA-mercapturate urine concentrations of two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples.


Subject(s)
Acetylcysteine/metabolism , Chromatography, Liquid/methods , Mass Spectrometry/methods , Thiotepa/metabolism , Calibration , Humans , Reproducibility of Results
17.
Arch Biochem Biophys ; 366(1): 89-94, 1999 Jun 01.
Article in English | MEDLINE | ID: mdl-10334868

ABSTRACT

Alkylating agents are extensively used in the treatment of cancer. The clinical usefulness of this class of anticancer drugs, however, is often limited by the emergence of drug-resistant tumor cells. Increased glutathione (GSH) conjugation through catalysis by GSH S-transferases (GSTs) is believed to be an important mechanism in tumor cell resistance to alkylating agents. In the present study, we report that the allelic variants of human Pi class GST (hGSTP1-1), which differ in their primary structures at amino acids in positions 104 and/or 113, exhibit significant differences in their activity in the GSH conjugation of alkylating anticancer drug thiotepa. Mass spectrometry revealed that the major product of the reaction between thiotepa and GSH was the monoglutathionyl-thiotepa conjugate. While nonenzymatic formation of monoglutathionyl-thiotepa was negligible, the formation of this conjugate was increased significantly in the presence of hGSTP1-1 protein. The hGSTP1-1-catalyzed GSH conjugation of thiotepa was time and protein dependent and followed Michaelis-Menten kinetics. The catalytic efficiency of hGSTP1-1(I104, A113) variant was approximately 1.9- and 2.6-fold higher compared with hGSTP1-1(V104,A113) and hGSTP1-1(V104,V113) isoforms, respectively. The results of the present study indicate that the hGSTP1-1 polymorphism may be an important factor in GST-mediated tumor cell resistance to thiotepa, and that subjects homozygous for the hGSTP1-1(I104,A113) allele, which is most frequent in human populations, are likely to be at a greater risk for developing GST-mediated resistance to thiotepa than heterozygotes or homozygotes with valine 104 background.


Subject(s)
Antineoplastic Agents, Alkylating/metabolism , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Glutathione/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Thiotepa/metabolism , Alleles , Genetic Variation , Glutathione S-Transferase pi , Humans , Inactivation, Metabolic , Kinetics
18.
Pharmacol Toxicol ; 84(3): 122-4, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10193672

ABSTRACT

This study was conducted to investigate if biotransformation of N,N,N-triethylene-thiophosphoramide (thio-TEPA) by liver slice incubations reflects the established gender pattern for rat and mouse. Liver slices from rat and mice of both genders were incubated with start concentrations of thio-TEPA of 5.2, 26, 52 and 104 microM for up to 240 min. Male rat liver slices eliminated thio-TEPA faster and formed more TEPA than female liver slices at any concentration. No gender difference was found for the elimination of thio-TEPA in mice, however, the female liver slices formed less TEPA than the male ones. Apparently female rat liver slices formed less TEPA than female mice liver slices. It is concluded that the liver slice incubation system in a robust manner reflects gender differences in rat drug biotransformation with special reference to thio-TEPA. It is also confirmed that these aspects of gender are less pronounced in the examined mouse species than in rats.


Subject(s)
Liver/metabolism , Thiotepa/metabolism , Animals , Female , In Vitro Techniques , Male , Mice , Rats , Rats, Sprague-Dawley , Sex Factors , Species Specificity , Time Factors
19.
Cancer Res ; 58(20): 4616-23, 1998 Oct 15.
Article in English | MEDLINE | ID: mdl-9788613

ABSTRACT

In this study, the role of glutathione S-transferase (GST) P1-1, the cellular reduced glutathione (GSH) status, and ATP-dependent efflux pumps in the cellular glutathione-dependent biotransformation of thiotepa and transport of the main metabolite monoglutathionylthiotepa in relation to cytotoxicity was studied in control and GST-P1-1-transfected MCF-7 cell lines. It was demonstrated that an enhanced cellular level of GST-P1-1 leads to an enhanced formation of monoglutathionylthiotepa, which is transported out of the cell into the medium. Monoglutathionylthiotepa was able to reversibly inhibit the activity of purified GST-P1-1, but only at nonphysiological concentrations, indicating that feedback inhibition of GST by its metabolites is not a relevant process in vivo. The GST activity, cellular GSH level, and/or ATP-dependent efflux of monoglutathionylthiotepa were modulated using ethacrynic acid, D,L-buthionine-S,R-sulfoximine, probenecid, and verapamil to understand the interplay between GSTs, glutathione conjugation, and efflux of glutathione conjugates in more detail. Inhibition of the GSH biosynthesis by D,L-buthionine-R,S-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, significantly reduced the glutathione conjugation of thiotepa and potentiated the cytotoxicity of thiotepa. Pretreatment of cells with ethacrynic acid resulted in decreased formation of monoglutathionylthiotepa as a result of inhibition of GST in the GST-P1-1 transfectant. In addition, the intracellular amount of monoglutathionylthiotepa increased in both of the cell lines on exposure to ethacrynic acid, indicating that transport of the glutathione conjugate was partially inhibited by the glutathione conjugate of ethacrynic acid. Transport activity of monoglutathionylthiotepa could also be inhibited by probenecid and verapamil, inhibitors of organic anion transport, without influencing the biotransformation capacity of the cells. It was demonstrated that inhibition of glutathione conjugate efflux by probenecid and verapamil leads to enhanced cytotoxicity, which indicates that besides thiotepa, monoglutathionylthiotepa is also cytotoxic for the cells. Only enhanced biotransformation and subsequent transport of the glutathione conjugate into the medium (which occurs with the GST-P1-1 transfectant) results in enhanced viability. Therefore, it was concluded that only enhanced biotransformation of thiotepa represents a real detoxification pathway when the resulting conjugate is transported out of the cells. Altogether, the results indicate that it is not the overexpression of GST per se but the interplay between GSH/GST and glutathione conjugate efflux pumps that results in increased resistance to alkylating anticancer drugs such as thiotepa.


Subject(s)
Antineoplastic Agents, Alkylating/metabolism , Breast Neoplasms/metabolism , Glutathione Transferase/physiology , Isoenzymes/physiology , Thiotepa/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , ATP-Binding Cassette Transporters/physiology , Biological Transport , Biotransformation , Cell Division/drug effects , Female , Glutathione S-Transferase pi , Humans , Multidrug Resistance-Associated Proteins , Thiotepa/pharmacology
20.
J Pharmacol Exp Ther ; 274(1): 270-5, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7616408

ABSTRACT

The anticancer drug and alkylating agent thiotepa is metabolized by oxidative desulfuration to yield the alkylating metabolite N,N',N"-triethylenephosphoramide (TEPA) in a reaction that is catalyzed by specific liver cytochrome P450 (CYP) enzymes, including CYP2B1, the major phenobarbital-inducible P450 of rat liver, and CYP2C11, a constitutively expressed, male-specific form. The present study investigates the potential for modulating the cytotoxicity and antitumor activity of thiotepa by prior treatment of tumor-bearing rats with the CYP2B1 inducer phenobarbital or the CYP2C11 inhibitor 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF-525A) and examines the role of TEPA in the cytotoxicity of thiotepa in vivo. Administration of thiotepa to adult male rats bearing 9L gliosarcoma, grown s.c., resulted in dose-dependent cytotoxicity (ED90 approximately 12 mg/kg i.v., single dose), as determined by a tumor excision/in vitro colony formation assay carried out 24 hr after drug treatment. Tumor growth delay experiments revealed that thiotepa (5 mg/kg) inhibited 9L tumor growth over a 5- to 7-day period after alkylating agent treatment and this effect was accompanied by moderate body weight loss. Pretreatment with phenobarbital, under conditions in which liver CYP2B1 levels and liver microsomal thiotepa desulfuration to yield TEPA are both markedly increased, did not alter thiotepa's short-term (24-hr) cytotoxicity, as judged by a tumor excision assay, nor did it affect the extent of bone marrow toxicity associated with drug treatment. However, phenobarbital did block the tumor growth delay effect of thiotepa and it also attenuated the body weight loss that occurred during the first 5 days after drug treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Brain Neoplasms/drug therapy , Cytochrome P-450 Enzyme System/metabolism , Gliosarcoma/drug therapy , Liver/enzymology , Thiotepa/therapeutic use , Animals , Bone Marrow/drug effects , Brain Neoplasms/pathology , Cell Division/drug effects , Dose-Response Relationship, Drug , Drug Synergism , Enzyme Induction , Gliosarcoma/pathology , Male , Phenobarbital/therapeutic use , Proadifen/pharmacology , Rats , Thiotepa/metabolism , Tumor Cells, Cultured
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