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1.
Oncotarget ; 5(7): 1761-9, 2014 04 15.
Article in English | MEDLINE | ID: mdl-24732092

ABSTRACT

Spores of some species of the strictly anaerobic bacteria Clostridium naturally target and partially lyse the hypoxic cores of tumors, which tend to be refractory to conventional therapies. The anti-tumor effect can be augmented by engineering strains to convert a non-toxic prodrug into a cytotoxic drug specifically at the tumor site by expressing a prodrug-converting enzyme (PCE). Safe doses of the favored prodrug CB1954 lead to peak concentrations of 6.3 µM in patient sera, but at these concentration(s) known nitroreductase (NTR) PCEs for this prodrug show low activity. Furthermore, efficacious and safe Clostridium strains that stably express a PCE have not been reported. Here we identify a novel nitroreductase from Neisseria meningitidis, NmeNTR, which is able to activate CB1954 at clinically-achievable serum concentrations. An NmeNTR expression cassette, which does not contain an antibiotic resistance marker, was stably localized to the chromosome of Clostridium sporogenes using a new integration method, and the strain was disabled for safety and containment by making it a uracil auxotroph. The efficacy of Clostridium-Directed Enzyme Prodrug Therapy (CDEPT) using this system was demonstrated in a mouse xenograft model of human colon carcinoma. Substantial tumor suppression was achieved, and several animals were cured. These encouraging data suggest that the novel enzyme and strain engineering approach represent a promising platform for the clinical development of CDEPT.


Subject(s)
Antineoplastic Agents/metabolism , Aziridines/metabolism , Biological Therapy , Carcinoma/therapy , Clostridium/enzymology , Colonic Neoplasms/therapy , Nitroreductases/metabolism , Spores, Bacterial/enzymology , Animals , Antineoplastic Agents/blood , Aziridines/blood , Biological Therapy/adverse effects , Clostridium/genetics , Mice , Neisseria meningitidis/enzymology , Neisseria meningitidis/genetics , Nitroreductases/genetics , Nitroreductases/isolation & purification , Organisms, Genetically Modified , Plasmids , Prodrugs/metabolism , Protein Engineering , Xenograft Model Antitumor Assays
2.
Rapid Commun Mass Spectrom ; 22(4): 462-70, 2008.
Article in English | MEDLINE | ID: mdl-18231986

ABSTRACT

A highly sensitive and selective liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to quantify the experimental anticancer agent EO9 and its metabolite EO5a in biological matrices. A 200-microL aliquot of human/dog plasma was spiked with a mixture of deuterated internal standards EO9-d3 and EO5a-d4 and extracted with 1.25 mL of ethyl acetate. Dried extracts were reconstituted in 0.1 M ammonium acetate/methanol (7:3, v/v) and 20-microL volumes were injected onto the LC system. Separation was achieved on a 150 x 2.1 mm C18 column using an alkaline eluent (1 mM ammonium hydroxide/methanol (gradient system)). The detection was performed by a Finnigan TSQ Quantum Ultra equipped with an electrospray ionization source operated in positive mode and enhanced mass resolution capability. It demonstrated improved sensitivity with a factor 10-20 for EO9 and EO5a over a 3-decades dynamic range, with acceptable accuracy and precision, when compared with the previously described assay for EO9 and EO5a, developed by our group, using an API 2000. The assay quantifies a range from 0.5 to 500 ng/mL for EO9 and EO5a using 200-microL human plasma and dog samples. The described mass resolution method was successfully applied for the evaluation of the pharmacokinetic profile of EO9 and its metabolite EO5a in human and dog plasma.


Subject(s)
Aziridines/blood , Blood Chemical Analysis/methods , Indolequinones/blood , Spectrometry, Mass, Electrospray Ionization/methods , Administration, Intravesical , Animals , Antineoplastic Agents/blood , Aziridines/administration & dosage , Dogs , Drug Evaluation/methods , Drug Evaluation, Preclinical/methods , Humans , Indolequinones/administration & dosage , Microchemistry/methods , Reproducibility of Results , Sensitivity and Specificity
3.
Toxicology ; 240(1-2): 70-85, 2007 Oct 30.
Article in English | MEDLINE | ID: mdl-17804142

ABSTRACT

5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954), a promising anti-tumour compound, is associated with clinical hepatotoxicity. We have previously demonstrated that human liver preparations are capable of endogenous 2- and 4-nitroreduction of CB 1954 to generate highly potent cytotoxins. The present study initially examined the in vitro metabolism of CB 1954 in S9 preparations of several non-clinical species and strains. The CD-1 nu/nu mouse and Sprague-Dawley rat were subsequently chosen for further assessment of in vivo metabolism and hepatotoxicity of CB 1954, as well as the mechanisms that may be involved. Animals were administered the maximum tolerated dose (MTD). At 562 micromol/kg, the mouse exhibited transaminase elevation and centrilobular hepatocyte injury. Moreover, thiol adducts as well as hepatic glutathione depletion paralleled temporally by maximal nitroreduction were observed. The rat had a much lower MTD of 40 micromol/kg and showed signs of gastro-intestinal disturbances. In contrast to mouse, peri-portal damage and biliary changes were observed in rat without any alterations in plasma biomarkers or hepatic glutathione levels. Immunohistochemical analysis did not reveal any correlation between the location of injury and expression of cytochrome P450 reductase and NAD(P)H quinone oxidoreductase 1, two enzymes implicated in the bioactivation of this drug. In conclusion, the present study showed that following administration of CB 1954 at the respective MTDs, hepatotoxicity was observed in both mouse and rat. However, the degree of sensitivity to the drug and the mechanisms of toxicity involved appear to be widely different between CD-1 nu/nu mice and Sprague-Dawley rats.


Subject(s)
Antineoplastic Agents , Aziridines , Chemical and Drug Induced Liver Injury/etiology , Microsomes, Liver/metabolism , Prodrugs , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/toxicity , Aziridines/blood , Aziridines/pharmacokinetics , Aziridines/toxicity , Chemical and Drug Induced Liver Injury/enzymology , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Dogs , Dose-Response Relationship, Drug , Female , Glutathione/metabolism , Humans , Male , Mice , Mice, Inbred Strains , Microsomes, Liver/drug effects , Microsomes, Liver/pathology , NAD(P)H Dehydrogenase (Quinone)/metabolism , NADPH-Ferrihemoprotein Reductase/metabolism , Prodrugs/pharmacokinetics , Prodrugs/toxicity , Rats , Rats, Sprague-Dawley , Species Specificity
4.
J Mass Spectrom ; 41(10): 1268-76, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16981212

ABSTRACT

A sensitive and specific LC-MS/MS assay for the quantitative determination of EO9 and its metabolite EO5a is presented. A 200-microl human plasma aliquot was spiked with a mixture of deuterated internal standards EO9-d3 and EO5a-d4 and extracted with 1.25 ml ethyl acetate. Dried extracts were reconstituted in 0.1 M ammonium acetate-methanol (7 : 3, v/v) and 25 microl-volumes were injected into the HPLC system. Separation was achieved on a 150 x 2.1 mm C18 column using an alkaline eluent (1 mM ammonium hydroxide-methanol (gradient system)). Detection was performed by positive ion electrospray followed by tandem mass spectrometry. The assay quantifies a range from 5 to 2500 ng/ml for EO9 and from 10 to 2500 ng/ml EO5a using 200 microl of human plasma samples. Validation results demonstrate that EO9 and EO5a concentrations can be accurately and precisely quantified in human plasma. This assay will be used to support clinical pharmacologic studies with EO9.


Subject(s)
Antineoplastic Agents, Alkylating/blood , Aziridines/blood , Indolequinones/blood , Calibration , Chromatography, High Pressure Liquid , Humans , Quality Control , Reference Standards , Reproducibility of Results , Spectrometry, Mass, Electrospray Ionization
5.
Cancer Chemother Pharmacol ; 58(4): 532-9, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16470409

ABSTRACT

OBJECTIVES: The objectives of this study were to characterize pharmacokinetics of N-2-chloroethylaziridine (CEA) in the rat model and assess the in vivo fraction of total clearance of phosphoramide mustard (PM) that furnished CEA to circulation. METHODS: The disposition of CEA was investigated following separate intravenous (iv) administrations of PM, synthetic CEA, and their combination to the Sprague-Dawley rats. In addition, in rats receiving prodrug cyclophosphamide (CP), plasma concentrations of CP and its metabolites, 4-hydroxycyclophosphamide (HOCP), PM, and CEA, were simultaneously quantified using GC/MS and stable isotope dilution techniques. RESULTS: Following iv administration of synthetic CEA, concentrations of CEA declined biexponentially with the mean terminal half-life and total body clearance of 47.5 min and 167 ml/min/kg, respectively. Urinary excretion of unchanged CEA was 0.164% of the administered dose. CEA was found to be the major circulating metabolite after iv administration of precursor PM to rats. The fraction of total clearance of PM that furnished CEA to circulation was estimated to be 100%, indicating virtually complete availability of the metabolite to circulation once formed. In rats administered with CP, PM exhibited the highest plasma and urinary concentrations compared to HOCP and CEA. CONCLUSIONS: For the first time, CEA was demonstrated to be an important in vivo metabolite of CP in the present study. In light of the poor permeability and in vivo stability of PM, the ultimate DNA alkylator, the findings obtained in this study suggested that CEA may contribute significantly to the overall antitumor activity of prodrug CP.


Subject(s)
Aziridines/pharmacokinetics , Phosphoramide Mustards/metabolism , Animals , Area Under Curve , Aziridines/blood , Gas Chromatography-Mass Spectrometry , Half-Life , Humans , Male , Metabolic Clearance Rate , Phosphoramide Mustards/pharmacokinetics , Protein Binding , Rats , Rats, Sprague-Dawley
6.
Clin Cancer Res ; 11(7): 2695-701, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15814651

ABSTRACT

PURPOSE: The purpose of our study was to investigate the cellular accumulation, DNA cross-linking ability, and cellular toxicity of RH1 (2,5-diaziridinyl-3-[hydroxymethyl[-6-methyl-1,4-benzoquinone), a novel DNA alkylating agent currently in clinical trials. In addition, the in vivo efficacy of RH1 formulated in different vehicles was also compared. EXPERIMENTAL DESIGN: RH1 is activated by the two-electron reducing enzyme NQO1 [NADPH:quinone oxidoreductase] forming a potent cytotoxic agent that cross-links DNA. We have used whole blood, cell lines, and primary explanted tumor cultures to measure both the cellular accumulation, DNA cross-linking, and cytotoxicity of RH1. Furthermore, the pharmacokinetic and pharmacodynamic characteristics of RH1 formulated in different vehicles were measured in vivo using the validated comet-X assay in mice bearing human tumor xenografts. RESULTS: Accumulation of RH1 was shown to be both time and concentration dependent, reaching a maximum after 2 hours and correlated well with DNA cross-linking measurements. DNA cross-linking in vitro could be detected at low (1-10 nmol/L) concentrations after as little as 2 hours exposure. In primary tumor cultures, RH1 induces much higher levels of DNA cross-links at lower doses than either mitomycin C or cisplatin. In vivo efficacy testing using polyvinyl pyrrolidone, saline, or cyclodextrin as vehicles showed DNA cross-links readily detectable in all tissues examined and was enhanced when given in cyclodextrin compared with polyvinyl pyrrolidone or saline. CONCLUSIONS: RH1 represents a potent bioreductive anticancer drug, which may prove effective in the treatment of cancers, particularly those that overexpress NQO1. DNA cross-linking can be reliably measured in tissue using the validated comet-X assay.


Subject(s)
Aziridines/pharmacology , Benzoquinones/pharmacology , Xenograft Model Antitumor Assays/methods , Animals , Aziridines/blood , Aziridines/pharmacokinetics , Benzoquinones/blood , Benzoquinones/pharmacokinetics , Cell Line, Tumor , Cell Proliferation/drug effects , Comet Assay/methods , Cross-Linking Reagents/pharmacology , DNA/chemistry , DNA/genetics , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Female , Humans , Mice , Mice, Nude , Treatment Outcome , Tritium
7.
Br J Pharmacol ; 137(5): 701-9, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12381684

ABSTRACT

The bioreductive drug EO9 (3-hydroxy-5-aziridinyl-1-methyl-2[indole-4,7-dione]-prop-beta-en-alpha-ol) has good pharmacodynamic properties in vitro, modest anti-tumour activity in experimental tumour models, but failed to show activity in clinical trials. Understanding the reasons for its poor efficacy in vivo is important in terms of progressing second generation analogues into the clinic. In two human tumour xenografts, direct intra-tumoural injection resulted in improved anti-tumour activity compared with intravenous administration suggesting that drug delivery to tumours is suboptimal. Compared with Mitomycin C (MMC) and the experimental agent MeDZQ, EO9 was rapidly cleared from the systemic circulation (t1/2=1.8 min) whereas MMC and MeDZQ had significantly increased plasma t1/2 values (14 and 22 min respectively). These three compounds demonstrated similar pharmacodynamic properties in terms of potency towards the NQO1 (NAD(P)H:Quinone oxidoreductase) rich H460 cell line in vitro but differed significantly in their in vivo activity with growth delays of 17.7, 4.5 and 1.0 days for MMC, MeDZQ and EO9 respectively. EO9 was rapidly metabolized by red blood cells in vitro (t1/2=14.5 min) which must contribute to its rapid pharmacokinetic elimination in vivo whereas MMC and MeDZQ were metabolized at comparatively slower rates (t1/2>120 min and 77.0 min respectively). In conclusion, the development of second generation EO9 analogues should address the issue of drug delivery and analysis of drug metabolism by murine whole blood in vitro could be utilized as a preliminary screen to identify lead compounds that are likely to have improved pharmacokinetic profiles in vivo.


Subject(s)
Aziridines/administration & dosage , Aziridines/chemistry , Indolequinones , Indoles/administration & dosage , Indoles/chemistry , Quinones/administration & dosage , Technology, Pharmaceutical/methods , Animals , Aziridines/blood , Drug Delivery Systems/methods , Humans , Indoles/blood , Mice , Mice, Nude , Quinones/blood , Quinones/chemistry , Tumor Cells, Cultured , Xenograft Model Antitumor Assays/methods
8.
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
9.
J Chromatogr B Biomed Sci Appl ; 731(2): 293-8, 1999 Aug 20.
Article in English | MEDLINE | ID: mdl-10510783

ABSTRACT

A high-performance liquid chromatography (HPLC) method is described for the measurement of the weak alkylating agent CB1954 in human plasma. CB1954 can be used as an innocuous prodrug designed for activation by bacterial nitroreductases in strategies of gene-directed enzyme-prodrug therapy, and becomes activated to a potent bifunctional alkylating agent. The HPLC method involves precipitation and solvent extraction and uses Mitomycin C (MMC) as an internal standard, with a retention time for MMC of 5.85 +/- 0.015 min, and for CB1954 of 10.72 +/- 0.063 min. The limit of detection for CB1954 is 2.9 ng/ml, and this compares favourably with systems involving direct analysis of plasma (limit of detection 600 ng/ml, approximately). The method is now being used for pharmacokinetic measurements in plasma samples from cancer patients entering phase I clinical trials of CB1954. Results using serial plasma samples from one patient are presented. The patient was treated intravenously with CB1954 (6 mg/m2), and plasma clearance of the drug showed biphasic kinetics with alpha half-life 14.6 min, and beta half-life 170.5 min.


Subject(s)
Antineoplastic Agents, Alkylating/blood , Aziridines/blood , Chromatography, High Pressure Liquid/methods , Antineoplastic Agents, Alkylating/pharmacokinetics , Aziridines/pharmacokinetics , Humans , Reference Standards , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry, Ultraviolet
10.
J Chromatogr B Biomed Sci Appl ; 729(1-2): 287-95, 1999 Jun 11.
Article in English | MEDLINE | ID: mdl-10410954

ABSTRACT

A HPLC assay and solid-phase extraction technique from human plasma has been developed and validated for the experimental anticancer agent, RH1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-1,4-benzoquinone) which is currently being evaluated by the CRC phase I/II committee. A 500 mg amino propyl solid-phase extraction cartridge was used to isolate RH1 from human plasma. Analysis was performed on a reversed-phase chromatography system using a 15 cm cyanopropyl column and isocratic elution with a 10% methanol-90% water (double distilled) solution. The lower limit of quantitation for RH1 was found to be 0.00375 microg/ml (3.75 ng/ml+/-8.3%) in water and following extraction from plasma. Recovery of >80%(+/-11.9%) was achieved over a five-day validation study. This method was used to carry out pre-clinical studies in BDF mice (standard strain of hybrid mice) at three dose levels (2, 5 and 10 mg/kg of RH1 in 0.9% (w/v) saline via an intraperotoneal injection). Standard Version of PC Winnonlin pharmacokinetic modelling software was used to model the data. A none-compartmental model was used to describe the disposition of RH1 in mice plasma. RH1 was rapidly eliminated from plasma with a mean plasma clearance of 23.4 ml/min, mean volume of distribution of 321.6 ml and mean t(1/2) alpha and beta decays of 4.8 and 9.6 min, respectively. RH1 in human and mouse whole blood and plasma was found to be stable up to 2 h.


Subject(s)
Antineoplastic Agents/blood , Aziridines/blood , Benzoquinones/blood , Chromatography, High Pressure Liquid/methods , Animals , Antineoplastic Agents/pharmacokinetics , Aziridines/pharmacokinetics , Benzoquinones/pharmacokinetics , Humans , Mice , Reproducibility of Results , Sensitivity and Specificity
11.
Eur J Cancer ; 32A(9): 1518-22, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8911111

ABSTRACT

EO9 is a synthetic indoloquinone which was designed to undergo redox cycling and formation of alkylating intermediates under bioreductive conditions. As part of a phase I clinical trial, EO9 plasma disposition was evaluated in 20 patients receiving 2.7-15 mg/m2i.v. weekly for 3 weeks. Pharmacokinetic studies were performed with the first and third dose of therapy and nine blood samples were obtained over 30 min postinfusion. Plasma EO9 was detected using HPLC UV and the disposition described by a two-compartment model. Wide variability in EO9 pharmacokinetics was observed. EO9 was rapidly eliminated from plasma with a median systemic clearance of 3.5 l/min/m2 (range 1.2-9.8), apparent volume of distribution of 6.2 l/m2 (1.0-34.9) and t 1/2 beta of 10.1 min (2.2-63.0). Substantial intrapatient variability was observed for all pharmacokinetic parameters. Linear regression and Bayesian methods were developed and validated for estimation of EO9 plasma AUC using up to three samples postinfusion. The use of two or three plasma samples provided precise estimation with acceptable prediction bias. In addition, a Bayesian algorithm offered more robust estimation of AUC and is preferable to linear regression models for future EO9 population pharmacokinetic analysis.


Subject(s)
Antineoplastic Agents/pharmacokinetics , Aziridines/pharmacokinetics , Indolequinones , Indoles/pharmacokinetics , Neoplasms/metabolism , Adult , Antineoplastic Agents/blood , Antineoplastic Agents/therapeutic use , Aziridines/blood , Aziridines/therapeutic use , Bayes Theorem , Female , Humans , Indoles/blood , Indoles/therapeutic use , Male , Metabolic Clearance Rate , Middle Aged , Neoplasms/drug therapy , Regression Analysis
12.
J Chromatogr B Biomed Appl ; 678(2): 219-25, 1996 Apr 12.
Article in English | MEDLINE | ID: mdl-8738025

ABSTRACT

A sensitive and specific method for the quantitative analysis of N-2-chloroethylaziridine (CEA), a volatile cytotoxic metabolite of cyclophosphamide, has been developed using gas chromatography-mass spectrometry and stable isotope dilution techniques. The high volatility problem of CEA during isolation procedure was overcome by the combined use of a deuterium-labeled analog as the internal standard and a Snyder column-concentrator assembly. The assay was found to be linear from 16.7 to 2667 ng/ml in rat plasma with a routine detection limit of 5 ng/ml. The within-run precision at 33, 333 and 1333 ng/ml (n = 6) was found to be 4.8, 4.9, and 6.1%, respectively. The between-run precision was 6.4% (n = 6). The dichloromethane extraction recoveries at 33, 333, and 1333 ng/ml were found to be 101, 98, and 91%, respectively (all at n = 6). However, the overall recovery through extraction and evaporation was only 18.3, 15.2, and 27.7% at 33, 333, and 1333 ng/ml levels, respectively. The analytical method was used to evaluate the generation of CEA from its precursors in sodium phosphate buffer, in cell culture media, and the degradation of CEA in these media. In pH 7.4, 0.067 M sodium phosphate buffer at 37 degrees C, both phosphoramide mustard (PM) and nornitrogen mustard (NNM) were degraded in an apparent first-order fashion with half-lives of 24.8 and 14.5 min, respectively. The generated CEA was rather stable in this buffer and degraded with a half-life of 20 h. It was found that 32% PM and 91% NNM were converted to CEA in pH 7.4, 0.067 M sodium phosphate buffer at 37 degrees C, respectively, and 41% PM was transformed into CEA in RPMI 1640 tissue culture media containing 10% FBS at 37 degrees C. The generated CEA was very stable in the culture media with a degradation half-life of 265 h.


Subject(s)
Antineoplastic Agents , Aziridines/blood , Cyclophosphamide/metabolism , Gas Chromatography-Mass Spectrometry/methods , Animals , Aziridines/chemistry , Buffers , Culture Media , Deuterium , Drug Stability , Gas Chromatography-Mass Spectrometry/statistics & numerical data , Half-Life , Hydrogen-Ion Concentration , Hydrolysis , Indicator Dilution Techniques , Kinetics , Methylene Chloride , Nitrogen Mustard Compounds/chemistry , Phosphoramide Mustards/chemistry , Rats , Sensitivity and Specificity , Volatilization
13.
Invest New Drugs ; 13(2): 175-6, 1995.
Article in English | MEDLINE | ID: mdl-8617583

ABSTRACT

Diaziquone (AZQ) is a lipid soluble alkylating agent which was designed for increased CNS penetration. Its principle toxicity is myelosuppression. We conducted a phase I trial using AZQ in combination with GM-CSF to determine if the maximal tolerate dose (MTD) of AZQ could be escalated. Using GM-CSF on a standard schedule, we were unable to escalate the previously determined MTD of diaziquone with the use of this colony stimulating factor.


Subject(s)
Antineoplastic Agents/pharmacokinetics , Aziridines/pharmacokinetics , Benzoquinones/pharmacokinetics , Bone Marrow/drug effects , Drugs, Investigational/pharmacokinetics , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacokinetics , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/blood , Antineoplastic Agents/therapeutic use , Aziridines/administration & dosage , Aziridines/blood , Aziridines/therapeutic use , Benzoquinones/administration & dosage , Benzoquinones/blood , Benzoquinones/therapeutic use , Blood Platelets/drug effects , Colorectal Neoplasms/drug therapy , Dose-Response Relationship, Drug , Drug Therapy, Combination , Drugs, Investigational/administration & dosage , Drugs, Investigational/therapeutic use , Granulocyte-Macrophage Colony-Stimulating Factor/administration & dosage , Granulocyte-Macrophage Colony-Stimulating Factor/blood , Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use , Humans , Infusions, Intravenous , Injections, Subcutaneous , Melanoma/drug therapy , Neutrophils/drug effects
14.
J Chromatogr ; 615(2): 309-15, 1993 Jun 02.
Article in English | MEDLINE | ID: mdl-8335709

ABSTRACT

A reversed-phase isocratic high-performance liquid chromatographic method is described for the simultaneous determination of EO9, 3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)prop-beta- en-alpha-ol (I), and its ring-opened aziridine analogue EO5A (II), employing ultraviolet detection. Solid-phase sample extraction was used without addition of an internal standard. Plots of peak heights and areas of I and II were linear in the range 5-10,000 ng/ml. The lower limit of detection of both I and II in plasma was 2 ng/ml. The between-day variation of I was 13.9% at 5 ng/ml and lower than 6.2% for concentrations > or = 10 ng/ml. The between-day variation of II at 5 ng/ml was 13.8% and lower than 4.5% for concentrations > or = 10 ng/ml. The assay was developed to enable pharmacological guiding of a phase I study of I in solid tumour cancer patients.


Subject(s)
Antineoplastic Agents/analysis , Aziridines/analysis , Indolequinones , Indoles/analysis , Antineoplastic Agents/blood , Antineoplastic Agents/urine , Aziridines/blood , Aziridines/urine , Chromatography, High Pressure Liquid , Humans , Indicators and Reagents , Indoles/blood , Indoles/urine , Spectrophotometry, Ultraviolet
15.
Eur J Cancer ; 29A(7): 1033-5, 1993.
Article in English | MEDLINE | ID: mdl-8499134

ABSTRACT

EO9[3-hydroxy-5-aziridinyl-1-methyl-2(1H-indole-4,7-dione)prop-bet a-en-alpha-ol] has been selected for phase I evaluation in Europe. Activity has been seen previously in a highly refractory, necrotic mouse adenocarcinoma (MAC 16) but EO9 is shown here to be inactive against early tumours (MAC 15A and MAC 13) and a well vascularised, well-differentiated established adenocarcinoma (MAC 26). EO9 becomes active against MAC 26 tumours when hydralazine (10 mg/kg) is administered 1 min after EO9. Co-administration of hydralazine decreases EO9 plasma clearance and increases plasma area under the curve values (0.053 to 0.115 micrograms h/ml). These pharmacokinetic changes are accompanied by anti-tumour activity but no increase in bone marrow toxicity so this therapeutic gain may be due, at least in part, to microenvironmental changes resulting from hydralazine induced tumour vascular shutdown.


Subject(s)
Antineoplastic Agents/therapeutic use , Aziridines/therapeutic use , Indolequinones , Indoles/therapeutic use , Adenocarcinoma/blood , Adenocarcinoma/drug therapy , Animals , Antineoplastic Agents/adverse effects , Antineoplastic Agents/blood , Aziridines/adverse effects , Aziridines/blood , Bone Marrow/drug effects , Colony-Forming Units Assay , Drug Screening Assays, Antitumor , Drug Synergism , Half-Life , Hydralazine/therapeutic use , Indoles/adverse effects , Indoles/blood , Mice
16.
Int J Radiat Oncol Biol Phys ; 22(4): 713-6, 1992.
Article in English | MEDLINE | ID: mdl-1544843

ABSTRACT

The indoloquinone EO9 is a novel and potent bioreductive agent related in structure to mitomycin C but differing in many aspects of its antitumor activity, toxicity, and enzymatic activation. Because it is about to undergo clinical trial, we have investigated the pharmacokinetics of EO9 in mice and rats. At the highest tolerated dose in male C3H/He mice (12 mg/kg iv) the initial plasma concentration (Co) was 1.8 micrograms/ml. The drug was cleared rapidly with a t1/2 of 1.9 min. The volume of distribution (Vd) was large (7.5 ml g-1) and the plasma clearance (Clp) correspondingly high (2.6 ml g-1 min-1). The AUCo-infinity was 4.8 micrograms ml-1 min. Equally rapid elimination was noted at the lower dose of 6 mg/kg iv. For comparison, the t1/2 for the same dose of mitomycin C was much longer at 16 min and the peak plasma level 4-fold higher. In male Sprague-Dawley rats receiving 3 mg kg-1 the Co was 1.5 micrograms/ml and the t1/2 was again short at 3.0 min. Vd was 2.2 ml g-1, Clp was 0.5 ml g-1 min-1, and AUCo-infinity was 6.2 micrograms ml-1 min. No parent drug was detected in urine, but extensive biotransformation was confirmed by the detection of around 20% of the dose as metabolites, including the aziridine ring-opened hydrolysis product EO5A. No drug or metabolite was detected in tumor or tissues. The results show that cytotoxic drug levels can be achieved for a short period in rodent plasma. The extremely fast excretion is consistent with the rapid rates of bioreductive metabolism in vitro. These data should be useful in the forthcoming clinical trials of EO9, where a pharmacokinetically guided dose escalation may be used, and also in the design and development of second generation analogues.


Subject(s)
Antineoplastic Agents/pharmacokinetics , Aziridines/pharmacokinetics , Indolequinones , Indoles/pharmacokinetics , Prodrugs/pharmacokinetics , Animals , Antineoplastic Agents/blood , Aziridines/blood , Cell Hypoxia , Indoles/blood , Male , Mice , Mice, Inbred C3H , Rats , Rats, Inbred Strains , Tissue Distribution
17.
J Chromatogr ; 532(2): 321-36, 1990 Nov 16.
Article in English | MEDLINE | ID: mdl-2084129

ABSTRACT

A high-performance liquid chromatographic method for the determination of the novel indoloquinone antitumour agent E09, 3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)prop-beta-e n-alpha - ol, in mouse plasma and urine is described. Following protein precipitation by means of methanol (2 volumes), separation and quantification of parent drug, metabolites and internal standard E012 (5-morpholine substituted analogue) were achieved on a 5-microns Resolve C18 Rad-Pak with a 15-min linear gradient of 10-30% acetonitrile in a 0.02 M pH 7.4 sodium phosphate buffer with UV detection at 280 and 310 nm. The utility of the assay is also demonstrated for the aziridine ring-opened analogue E05A. 3-hydroxymethyl-5-beta-hydroxyethylamino-2-(1H-indole-4,7-dione)pr op-beta-en- alpha-ol. Plots of area ratios of analytes versus internal standard were linear in the range 50-15,000 ng/ml. The detection limit for indoloquinones in plasma was ca. 30 ng/ml. The within-assay and day-to-day variation were consistently lower than 12.5%. The assay was applied in preliminary pharmacokinetic investigations. One minor metabolite of E09 could be identified; further metabolites were characterized by ultraviolet-visible spectra.


Subject(s)
Antineoplastic Agents/analysis , Aziridines/analysis , Chromatography, High Pressure Liquid/methods , Indolequinones , Indoles/analysis , Quinones/analysis , Animals , Antineoplastic Agents/pharmacokinetics , Aziridines/blood , Aziridines/urine , Chromatography, High Pressure Liquid/statistics & numerical data , Half-Life , Indoles/blood , Indoles/urine , Magnetic Resonance Spectroscopy , Male , Mice , Mice, Inbred C3H , Quinones/blood , Quinones/urine , Spectrophotometry, Ultraviolet
18.
Eur J Cancer ; 26(2): 107-12, 1990 Feb.
Article in English | MEDLINE | ID: mdl-2138904

ABSTRACT

Plasma levels of 2,5-diaziridinyl-3,6-bis(2-hydroxyethylamino)-1,4-benzoquinone (BZQ, NSC 224070) were measured in nine patients after i.v. administration of the drug during a Phase I trial. Our own isocratic high performance liquid chromatographic (HPLC) method with a sensitivity of 3 ng/ml was used to quantify BZQ. Patients receiving 18-60 mg BZQ i.v. showed alpha and beta plasma decays with half-lives of 6.2 +/- 1.5 (mean +/- S.D.) and 24 +/- 4 min respectively. The apparent volume of the central compartment was 12.2 +/- 4.6 l, and the total volume of distribution was 33.6 +/- 11.3 l. The calculated plasma AUCs were linearly related to dose. A marked similarity in kinetic parameters was found for BZQ and diaziquone (AZQ, NSC 182986), another diaziridinylbenzoquinone that has recently completed phase II clinical trials.


Subject(s)
Antineoplastic Agents/pharmacokinetics , Aziridines/pharmacokinetics , Benzoquinones , Aged , Antineoplastic Agents/blood , Aziridines/blood , Chromatography, High Pressure Liquid , Drug Evaluation , Female , Humans , Male , Middle Aged
20.
Blood ; 67(1): 182-7, 1986 Jan.
Article in English | MEDLINE | ID: mdl-3940546

ABSTRACT

Diaziquone given as a bolus has not been effective in patients with relapsed or refractory leukemia. Because of in vitro data suggesting enhancement of diaziquone-induced cytotoxicity for human and murine leukemia cells with increased duration of drug exposure and the relatively short terminal plasma half-life of diaziquone, 49 patients (34 acute nonlymphocytic leukemia [ANLL], six chronic myelogenous leukemia in blast crisis [CML-B], five acute lymphocytic leukemia [ALL], four 2 degrees ANLL) with leukemia were given diaziquone as a continuous infusion for seven days. The maximum tolerated dose was 28 mg/m2/d for seven days. The dose-limiting toxicity was the duration of bone marrow aplasia (median, 49 days to greater than 500 PMNs in responders; range, 28 to 101 days). Nonhematologic toxicity was minimal. Responses occurred only in patients with relapsed ANLL, of whom 26 were treated at effective doses. There were six complete responses (CR) (23%) and two partial responses (PR) (8%), although five of eight responders never achieved platelet counts greater than 100,000/microL. Thrombocytopenia in these patients was felt to be a manifestation of diaziquone effect, not persistence of leukemia. The median duration of CR was 195 days (range, 88 to 860+). One patient had active CNS leukemia at the start of treatment and has had a durable (28+ month) CR in both sites of disease. Diaziquone produced prolonged aplasia in patients with secondary ANLL and CML-B (five of ten patients died aplastic), whereas patients with ALL all had regrowth of leukemia and two failed to become aplastic. The lack of significant nonhematologic toxicity and the activity in patients with relapsed ANLL render diaziquone of interest as second-line therapy or consolidation therapy in first remission for patients with ANLL.


Subject(s)
Antineoplastic Agents/administration & dosage , Aziridines/administration & dosage , Azirines/administration & dosage , Benzoquinones , Leukemia/drug therapy , Acute Disease , Adolescent , Adult , Aged , Antineoplastic Agents/adverse effects , Antineoplastic Agents/blood , Aziridines/adverse effects , Aziridines/blood , Female , Humans , Kinetics , Male , Middle Aged , Platelet Count , Recurrence
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