Modulating effects of naturally occurring indoles on SCE induction depend largely on the type of mutagen

The modulating effects of pretreatment of cultured cells with indole-3-carbinol (I3C) and indole-3-acetonitrile (I3A) on the induction of sister-chromatid exchanges (SCEs) by mutagens from different chemical classes were investigated. Cultured primary chick embryo hepatocytes were treated for different periods with I3C (25 micrograms/ml) and with I3A (35 micrograms/ml). Treatment with I3C resulted in a 3-fold increase in ethoxyresorufine-O-deethylase (Erod) activity and a 2-fold increase in ethoxycoumarine-O-deethylase (Etco) activity. Treatment with I3A resulted in a 1.6-fold increase in Erod activity and a 2-fold increase in Etco activity. Pretreatment of cultured primary chick embryo hepatocytes with I3C resulted in a 30-45% decrease in the number of SCEs induced by benzo[a]pyrene (B(a)P) and dimethylnitrosamine (DMN) in co-cultured V79 Chinese hamster cells. No decrease in SCE induction was observed for 2-aminoanthracene (2AA) and the direct-acting alkylating agent ethyl methanesulphonate (EMS). In contrast, when dibromoethane (DBE) was tested pretreatment with I3C resulted in an increase in SCE induction. Pretreatment with I3A again resulted in a 20-40% decrease in SCE induction for B(a)P whereas no decrease was observed for DMN, 2AA and EMS. The results of this study indicate that the type of effect of indole pretreatment largely depends on the type of mutagen selected.     

Naturally Occurring Ah-Receptor Agonists in Foods: Implications Regarding Dietary Dioxin Exposure and Health Risk

The human diet contains numerous naturally-occurring compounds that are aryl hydrocarbon receptor (AhR) agonists. This analysis compares the dietary TCDD-equivalent (TEQ) dose from specific vegetable indoles vs. the AhR-active PCDD/Fs. Daily dietary doses of indole-3-carbinole (I3C) and its condensation product indolo [3,2-b]carbazole (ICZ) were derived from the published literature. Relative estimate of potency (REP) values were developed for I3C (8.7 × 10^?7) and ICZ (0.5). The TEQ doses of I3C and ICZ together comprised >99% of the total daily TEQ dose; the daily ICZ TEQ dose (1.4 × 10⁶ pg TEQ/day) was approximately 45,000-fold greater than the current dietary PCDD/F TEQ dose (32 pg TEQ/day). When 30-year accumulated body burden and area-under-the curve doses were calculated, I3C/ICZ still comprised a significant fraction (up to 95 and 96%, respectively) of the total TEQ dose. Further, reduction or elimination of meat and dairy products yielded a minimal (less than 4%) decrease in total TEQ dose. These findings indicate that reducing the intake of animal products (the primary source of dietary PCDD/Fs) might not achieve a significant reduction in total “dietary dioxin TEQ” dose; the comparisons also suggest that trace levels of PCDD/Fs in the human diet are unlikely to pose a significant health risk.     

Cytotoxicity and DNA topoisomerase inhibitory activity of benz[f]indole-4,9-dione analogs

A series of benz[f]indole-4,9-diones, based on the antitumor activity of 1,4-naphthoquinone, were synthesized and evaluated for their cytotoxic activity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer), and also for the inhibition of human DNA topoisomerases I and II activity in vitro. Several compounds including 2-amino-3-ethoxycarbonyl-N-methyl-benz[f]indole-4,9-dione showed a potential cytotoxic activity judged by IC50<20.0 microg/ml in the panel of cancer cell lines. Especially, 2-hydroxy-3-ethoxycarbonyl-N-(3,4-dimethylphenyl)-benz[f]indole-4,9-dione had potential selective cytotoxicity against lung cancer cells (IC50=0.4 microg/ml)) compared to colon (IC50>20.0 microg/ml) and stomach (IC50>20.0 microg/ml) cancer cells. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities were used. In a topoisomerase I-mediated relaxation assay using human placenta DNA topoisomerase I and supercoiled pHOTI plasmid DNA, 2-amino-3-ethoxycarbonyl-N-(4-fluorophenyl)-benz[f]indole-4,9-dione had the most potent inhibitory activity among the compounds tested. However, most of the compounds showed only weak inhibition of the DNA topoisomerase II-mediated KDNA (Kinetoplast DNA) decatenation assay, except for 2-amino-3-ethoxycarbonyl-N-(4-methylphenyl)-benz[f]indole-4,9-dione and 2-amino-3-ethoxycarbonyl-N-(2-bromoehtyl)-benz[f]indole-4,9-dione with a moderate inhibitory activity. These results suggest that several active compounds had relatively selective inhibitory activity against toposiomearse I compared to toposiomerase II. No obvious correlation was observed between the cytotoxicity of the individual compound and the inhibitory activity of DNA relaxation and decatenation by topoisomerase I and II, respectively, in vitro.     

Structure elucidation of acid reaction products of indole-3-carbinol: Detection in vivo and enzyme induction in vitro

The potency of indole-3-carbinol (I3C) to form condensation products under acidic aqueous conditions was studied. After identifying a known dimer, 3,3′-diindolylmethane (DIM), we elucidated the structures of two trimers also found in acid reaction mixtures: 5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b′:7,8-b″]tri-indole (CTI), and 2,3-bis[3-indolylmethyl] indole (BII). The formation of these indole oligomers was shown to be pH dependent. The highest amounts of DIM and BII were formed in aqueous solutions having a pH value ranging from 4 to 5. No CTI could be detected at pH values above 4.5. In rats that received an oral dose of I3C we could detect DIM and BII in gastric contents, stomach tissue, small intestine and liver. No CTI could be detected in vivo after oral exposure to I3C. In in vitro experiments, using rat hepatocytes, the cytochrome P-450IA1 apoprotein level, 7-ethoxyresorufin O-deethylation activity (EROD) and DT-diaphorase activity (DTD) were markedly enhanced by DIM and CTI as well as BII.     

Cytotoxicity and DNA Topoisomerase Inhibitory Activity of Benz[f]indole-4,9-dione Analogs

A series of benz[f]indole-4,9-diones, based on the antitumor activity of 1,4-naphthoquinone, were synthesized and evaluated for their cytotoxic activity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer), and also for the inhibition of human DNA topoisomerases I and II activity in vitro. Several compounds including 2-amino-3-ethoxycarbonyl-N-methyl-benz[f]indole-4,9-dione showed a potential cytotoxic activity judged by IC₅₀<20.0 μg/ml in the panel of cancer cell lines. Especially, 2-hydroxy-3-ethoxycarbonyl-N-(3,4-dimethylphenyl)-benz[f]indole-4,9-dione had potential selective cytotoxicity against lung cancer cells (IC₅₀=0.4 μg/ml)) compared to colon (IC₅₀>20.0 μg/ml) and stomach (IC₅₀>20.0 μg/ml) cancer cells. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities were used. In a topoisomerase I-mediated relaxation assay using human placenta DNA topoisomerase I and supercoiled pHOTI plasmid DNA, 2-amino-3-ethoxycarbonyl-N-(4-fluorophenyl)-benz[f]indole-4,9-dione had the most potent inhibitory activity among the compounds tested. However, most of the compounds showed only weak inhibition of the DNA topoisomerase II-mediated KDNA (Kinetoplast DNA) decatenation assay, except for 2-amino-3-ethoxycarbonyl-N-(4-methylphenyl)-benz[f]indole-4,9-dione and 2-amino-3-ethoxycarbonyl-N-(2-bromoehtyl)-benz[f]indole-4,9-dione with a moderate inhibitory activity. These results suggest that several active compounds had relatively selective inhibitory activity against toposiomearse I compared to toposiomerase II. No obvious correlation was observed between the cytotoxicity of the individual compound and the inhibitory activity of DNA relaxation and decatenation by topoisomerase I and II, respectively, in vitro.     

Inhibition of premature death by isothiocyanates through immune restoration in LP-BM5 leukemia retrovirus-infected C57BL/6 mice

The purpose of this study was to determine the effect of isothiocyanates (ITCs) in delaying the progression of the murine immunodeficiency virus to murine AIDS, resulting in increased life span. Furthermore, we investigated the role of ITCs in modulating immune dysfunction caused by LP-BM5 retrovirus infection. Among the tested ITCs, oral administration of sulforaphane (SUL), benzyl isothiocyante (BITC), and phenethyl isothiocyanate (PEITC) showed the inhibition of premature death caused by LP-BM5 retrovirus infection, while indolo[3,2-b] carbazole (ICZ) and indole-3-carbinol (I3C) did not delay the progress of the LP-BM5 retrovirus to murine AIDS. Inhibition of premature death by BITC, PEITC, and SUL could be explained by restoration of the immune system and down regulation of free radicals. Dysfunction of T and B cell mitogenesis caused by retrovirus infection in primary cultured splenocytes has been partially recovered with administration of BITC, PEITC, and SUL. There was a shift from imbalanced cytokine production (increased Th2 and decreased Th1 cell cytokine production) into balanced Th1/Th2 cell secretion of cytokines under administration of these ITCs during the development of murine AIDS. Hepatic vitamin E level was significantly restored by administration of these ITCs, in accordance with reduced hepatic lipid peroxidation levels. This study suggests that certain types of ITCs have beneficial effects in preventing premature death during progression to murine AIDS by restoration of immune dysfunction and removal of excessive free radicals, implying that selective usage of ITCs would be helpful in retarding the progression from HIV infection to AIDS.     

Inhibition of Premature Death by Isothiocyanates through Immune Restoration in LP-BM5 Leukemia Retrovirus-Infected C57BL/6 Mice

The purpose of this study was to determine the effect of isothiocyanates (ITCs) in delaying the progression of the murine immunodeficiency virus to murine AIDS, resulting in increased life span. Furthermore, we investigated the role of ITCs in modulating immune dysfunction caused by LP-BM5 retrovirus infection. Among the tested ITCs, oral administration of sulforaphane (SUL), benzyl isothiocyante (BITC), and phenethyl isothiocyanate (PEITC) showed the inhibition of premature death caused by LP-BM5 retrovirus infection, while indolo[3,2-b] carbazole (ICZ) and indole-3-carbinol (I3C) did not delay the progress of the LP-BM5 retrovirus to murine AIDS. Inhibition of premature death by BITC, PEITC, and SUL could be explained by restoration of the immune system and down regulation of free radicals. Dysfunction of T and B cell mitogenesis caused by retrovirus infection in primary cultured splenocytes has been partially recovered with administration of BITC, PEITC, and SUL. There was a shift from imbalanced cytokine production (increased Th2 and decreased Th1 cell cytokine production) into balanced Th1/Th2 cell secretion of cytokines under administration of these ITCs during the development of murine AIDS. Hepatic vitamin E level was significantly restored by administration of these ITCs, in accordance with reduced hepatic lipid peroxidation levels. This study suggests that certain types of ITCs have beneficial effects in preventing premature death during progression to murine AIDS by restoration of immune dysfunction and removal of excessive free radicals, implying that selective usage of ITCs would be helpful in retarding the progression from HIV infection to AIDS.     

Biosynthesis and metabolism of indole-3-ethanol and indole-3-acetic acid by Pinus sylvestris L. needles

Combined gas chromatography-mass spectrometry has been used to identify indole-3-ethanol (IEt) in a purified extract from needles of Pinus sylvestris L. Quantitative estimates obtained by high-performance liquid chromatography with fluorescence detection, corrected for samples losses occurring during purification, indicate that Pinus needles contain 46±4 ng g^-1 IEt. This compares with 24.5±6.5 ng g^-1 indole-3-acetic acid (IAA) and 2.3±0.4 ng g^-1 indole-3-carboxylic acid (ICA) (Sandberg et al. 1984, Phytochemistry, 23, 99–102). Metabolism studies with needles incubated in a culture medium in darkness revealed that both [3-¹⁴C]-tryptophan and [2-¹⁴C]tryptamine mine are converted to [¹⁴C]IEt. It was also shown that [3-¹⁴C]IEt acted as a precursor of [¹⁴C]IAA. The observed metabolism appears to be enzymic in nature. The [2-¹⁴C]IAA was not catabolised to [¹⁴C]ICA in detectable quantities implying that, at best, only a minor portion of the endogenous ICA pool in the Pinus needles originates from IAA.Abbreviations DEAE diethylaminoethyl - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - ICA indole-3-carboxylic acid - IEt indole-3-ethanol - PVP polyvinylpyrrolidone     

Validation of liquid chromatography assay for the quantitation of (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]propionic acid (SU006668) in human plasma and its application to a phase I clinical trial

The validation of an analytical method to quantify the antiangiogenic, (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]propionic acid (SU006668) for pharmacokinetic determination in a phase I clinical trial, is described. HPLC, with a gradient mobile phase and UV detection at 440 nm, was used. SU006668 was extracted from plasma by precipitation of proteins with acetonitrile. The assay was linear from 25 to 2000 ng/ml (r(2)=0.997); sensitive (limit of quantification 25 ng/ml), accurate (RE 2.6-11.9%) and reproducible (inter-batch precision C.V. 3.2%). Pharmacokinetic data for six patients are presented. They show linear pharmacokinetics with a low volume of distribution and induction at doses of 50, 100 and 200 mg/m(2).     

3,3′-Diindolylmethane but not indole-3-carbinol activates Nrf2 and induces Nrf2 target gene expression in cultured murine fibroblasts

Abstract

There is increasing interest in the gene-regulatory activity of Brassica vegetable derived phytochemicals such as 3,3′-diindolylmethane (DIM) and indole-3-carbinol (I3C). DIM is formed under acidic conditions by dimerization of I3C. This study compared the Nrf2 activating potential of DIM and I3C in murine fibroblasts (NIH3T3). In contrast to its precursor I3C, DIM induces the transactivation of Nrf2. Furthermore, Nrf2 targets such as HO-1, γGCS and NQO1 were increased on the mRNA and protein levels following DIM treatment. DIM was less potent than sulforaphane (used as positive control) in inducing Nrf2-dependent gene expression. The present data suggest that the dimerization of I3C to DIM increases its Nrf2 inducing activity.     

High-performance liquid chromatographic method for the determination of a novel thymidylate synthase inhibitor, AG 331, in human serum

AG 331 is a novel thymidylate synthase inhibitor currently in Phase I clinical trial. To determine the pharmacokinetic parameters of AG 331 in human subjects, a suitable analytical method was developed using high-performance liquid chromatography. Serum and urine samples were prepared using both solid-phase extraction and solvent extraction. Either 4,4′-diaminodiphenyl sulfone or benz[cd]indole-2(1H)-one were used as internal standards for the method. A reversed-phase C₁₈ analytical column completely resolved the drug and internal standard peaks from non-specific substances present in biological matrix. The method was validated for precision, accuracy, and reproducibility in serum and was linear over a concentration range of 50–2000 ng/ml, with a limit of detection of 20.0 ng/ml and a quantifiable limit of 50 ng/ml.     

3,3'-Diindolylmethane but not indole-3-carbinol activates Nrf2 and induces Nrf2 target gene expression in cultured murine fibroblasts

There is increasing interest in the gene-regulatory activity of Brassica vegetable derived phytochemicals such as 3,3'-diindolylmethane (DIM) and indole-3-carbinol (I3C). DIM is formed under acidic conditions by dimerization of I3C. This study compared the Nrf2 activating potential of DIM and I3C in murine fibroblasts (NIH3T3). In contrast to its precursor I3C, DIM induces the transactivation of Nrf2. Furthermore, Nrf2 targets such as HO-1, γGCS and NQO1 were increased on the mRNA and protein levels following DIM treatment. DIM was less potent than sulforaphane (used as positive control) in inducing Nrf2-dependent gene expression. The present data suggest that the dimerization of I3C to DIM increases its Nrf2 inducing activity.     

Simultaneous determination of nimesulide and hydroxynimesulide in rat plasma,cerebrospinal fluid and brain by liquid chromatography using solid-phase extraction

A liquid chromatographic method with UV detection for the quantification of nimesulide (N) and hydroxynimesulide (M1) in rat plasma, cerebrospinal fluid (CSF) and brain tissue is reported. Plasma samples (250 microl) and brain homogenates added with the right amount of the internal standard (I.S., 2'-(cyclohexyloxy)-4'-nitrophenyl methanesulphonanilide, NS398) are extracted on C(18) disposable cartridges by solid-phase extraction (SPE), while CSF samples are analyzed without any extraction. The separation is performed at room temperature on a Waters Symmetry C(18) 3.5 microm (150x4.6 mm I.D.) column with acetonitrile-sodium citrate buffer pH 3.00 (53:47, v/v) as mobile phase, at a flow-rate of 1.1 ml/min and detection at 240 nm. The retention times are 3.3, 6.0 and 9.9 min for M1, N and I.S., respectively. The lower limits of quantitation for either nimesulide and M1 are 25 ng/ml for plasma, 20 ng/ml for CSF and 25 ng/g for brain tissue. The calibration curves are linear up to 10,000 ng/ml for plasma, 5000 ng/ml for CSF and 5000 ng/g for brain tissue. This new assay can be applied to the study of the role of nimesulide in the modulation of neuroinflammatory processes.     

Specificity of insulin-like growth factor binding to type-II IGF receptors in rabbit mammary gland and hypophysectomized rat liver

We have reevaluated IGF binding specificity to membrane receptors in rabbit mammary gland (RMG) and hypophysectomized rat liver (HRL) using recombinant DNA-derived and synthetic analogues of human IGF-I and highly purified IGF-II. SDS-PAGE demonstrated that [125I]IGF-I bound to type-I IGF receptors in RMG; this binding was inhibited in a similar fashion by the IGF-I analogues (IC50 = 10 ng/ml) and to a lesser extent by IGF-II (IC50 = 60 ng/ml). [125I]IGF-II bound to type-II IGF receptors in both RMG and HRL. The IC50 for IGF-II was 9 and 3 ng/ml with RMG and HRL, respectively. At a dose as high as 1 microgram/ml, IGF-I analogues inhibited less than 20% of [125I]IGF-II binding. These results suggest that IGF-I has little or no affinity for type-II IGF receptors.     

Ultraviolet radiation-induced non-melanoma skin cancer in the Crl:SKH1:hr-BR hairless mouse: augmentation of tumor multiplicity by chlorophyllin and protection by indole-3-carbinol.

Over 1 million new cases of ultraviolet radiation-induced non-melanoma skin cancers (NMSC) per year now occur in the USA and the incidence of these diseases continues to increase. New preventative strategies are required. The hypothesis tested was that dietary administration of the putative cancer chemopreventatives sodium-copper-chlorophyllin (Chlor) or indole-3-carbinol (I3C) would inhibit UV-induced skin carcinogenesis in the Crl:SKH1:hr-BR hairless mouse. Groups of 20 mice were pre-fed isocaloric/isonutritive 20% corn-oil AIN-76a based diets that contained either Chlor (1.52 g%), I3C (5.08 g%) or no chemopreventative (control) for 2 weeks followed by exposure of their dorsal skin to a 10 week incremental, sub-erythemal, carcinogenic simulated solar UV exposure regime. Feeding was continued for the duration of the experiment. Matched non-UV exposed dietary groups were also included in the experimental design. The diets had no significant (p > 0.05) effect on body weight, feed consumption, cutaneous methanol-extractable UV photoprotective substances or on cutaneous UV-reflective characteristics. By day 180, UV-irradiated mice fed the Chlor had a significantly (p < 0.05) higher tumor multiplicity (33.6 +/- 4.72; mean +/- SEM) than UV-irradiated control animals (22.8 +/- 4.25). UV-irradiated mice fed I3C had a significantly (p < 0.001) lower tumor multiplicity (13.0 +/- 2.42) than that of both the UV-irradiated control and UV-irradiated Chlor-fed mice. The Chlor or I3C diets did not significantly (p > 0.05) affect UV-induced systemic suppression of contact hypersensitivity responses. These results demonstrate augmentation of the UV-induced cutaneous carcinogenic process by dietary chlorophyllin and protection from this carcinogenic process by indole-3-carbinol via mechanisms that do not involve changes in skin optical properties, modulation of photoimmunosuppression or caloric/nutrient effects.     

Effects of dietary chemopreventive phytochemicals on P-glycoprotein function

The effects of dietary phytochemicals on P-glycoprotein function were investigated using human multidrug-resistant carcinoma KB-C2 cells and the fluorescent P-glycoprotein substrates daunorubicin and rhodamine 123. The effects of natural chemopreventive compounds, capsaicin found in chilli peppers, curcumin in turmeric, [6]-gingerol in ginger, resveratrol in grapes, sulforaphane in broccoli, 6-methylsulfinyl hexyl isothiocyanate (6-HITC) in Japanese horseradish wasabi, indole-3-carbinol (I3C) in cabbage, and diallyl sulfide and diallyl trisulfide in garlic, were examined. The accumulation of daunorubicin in KB-C2 cells increased in the presence of capsaicin, curcumin, [6]-gingerol, and resveratrol in a concentration-dependent manner. The accumulation of rhodamine 123 in KB-C2 cells was also increased, and the efflux of rhodamine 123 from KB-C2 cells was decreased by these phytochemicals. Sulforaphane, 6-HITC, I3C, and diallyl sulfide and diallyl trisulfide had no effect. These results suggest that dietary phytochemicals, such as capsaicin, curcumin, [6]-gingerol, and resveratrol, have inhibitory effects on P-glycoprotein and potencies to cause drug-food interactions.     

Determination of clozapine and its major metabolites in human plasma and red blood cells by high-performance liquid chromatography with ultraviolet absorbance detection

An isocratic high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of clozapine (8-chloro-11-(4′-methyl)piperazino-5H-dibenzo[b,e]-1,4-diazepine) and its two major metabolites in plasma and red blood cells (RBCs). The method involves sample clean-up by liquid-liquid extraction with ethyl acetate. The organic phase was back-extracted with 0.1 M hydrochloric acid. Loxapine served as the internal standard. The analytes were separated by HPLC on a Kromasil Ultrabas C₁₈ analytical column (5 μm particle size; 250×4.6 mm I.D.) using acetonitrile-phosphate buffer pH 7.0 (48:52, v/v) as eluent and were measured by UV absorbance detection at 254 nm. The limits of quantification were 20 ng/ml for clozapine and N-desmethylclozapine and 30 ng/ml for clozapine N-oxide. Recovery from plasma or RBCs proved to be higher than 62%. Precision, expressed as % C.V., was in the range 0.6–15%. Accuracy ranged from 96 to 105%. The method's ability to quantify clozapine and two major metabolites simultaneously with precision, accuracy and sensitivity makes it useful in therapeutic drug monitoring.     

Determination of 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiophen-2-yl]cyclopent-3-enecarboxylic acid (CP-191,166), an angiotensin II antagonist, in dog and rat plasma by high-performance liquid chromatography

A simple and precise high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of a novel angiotensin II antagonist, 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiopen-2-yl)cyclopent-3-enecarboxylic acid (CP-191,166, I), in dog and rat plasma. The internal standard (II, a saturated derivative of I) and analyte were extracted by liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Zorbax C₈ narrow-bore column with ultraviolet detection at 289 nm. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01–10.0 μg/ml (r²>0.99). In dog and rat plasma, intra- and inter-assay precision ranged from 0.00 to 3.36% and 0.00 to 4.95%, respectively. The average recoveries were similar (73%) for both I and II and the upper limit of quantification of I can be as high as 500 μg/ml. The method described has been successfully applied to the quantification of I in about 2000 dog and rat plasma samples over a nine-month period.     

Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg6, d-Trp7,9, mePhe8]-substance P (6–11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate

[Arg⁶, d-Trp^7,9, mePhe⁸]-substance P (6–11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met¹¹ (M2) and G[Met¹¹(O)] (M3). Gradient elution was employed using 40 mM ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (μBondapak C₁₈, 30 cm×2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40°C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G–P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1–3 and G–P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1–3. Sample clean-up by solid-phase extraction using C₂-bonded 40 μm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml–100 μg/ml) normally varied by <10%, although at the highest concentrations of M1 and M2 studied (50 μg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.