Rapid and sensitive quantitation of the antiproliferative agent mitoguazone in small volumes of plasma by high-performance liquid chromatography with ultraviolet detection.

Mitoguazone is an antiproliferative agent used in chemotherapy. This study describes a simple and sensitive high-performance liquid chromatographic method for the determination of mitoguazone in 100 microl of plasma. Samples were deproteinized with 100 microl of a solution of internal standard (amiloride, 10 microg/ml) in acetonitrile. An aliquot of the supernatant was injected onto the column. HPLC separation was achieved on a silica column with the mobile phase of methanol-50 mM potassium phosphate buffer (pH 3)-triethylamine (80:20:0.3, v/v), at a flow-rate of 1 ml/min. The eluent was detected at 320 nm. The retention time was about 5.5 min for amiloride and 12 min for mitoguazone. No endogenous substances were found to interfere. Calibration curves were linear from 0.25 to 50 microg/ml. The absolute recoveries of mitoguazone and amiloride were both greater than 84%. The limit of quantitation was 0.25 microg/ml. The intra- and inter-day precision (expressed as RSD) was 5.8%, or less, and the accuracy was 94.7% of the nominal concentration. The method is suitable in pharmacokinetic investigation and monitoring mitoguazone concentration.

The use of capillary electrophoresis as part of a specificity testing strategy for mitoguazone dihydrochloride HPLC methods.

Capillary electrophoresis (CE) has been used as part of a validation experiment designed to prove the specificity of high performance liquid chromatography (HPLC) methods used for analysis of mitoguazone dihydrochloride drug substance. Data regarding accuracy, precision and sensitivity of the CE methods are presented as well as a comparison of results obtained from CE, HPLC and thin-layer chromatography (TLC) analysis of samples stressed under a variety of conditions. It was concluded that, not only were the HPLC methods being investigated specific, but that CE could potentially be used to replace HPLC for the routine assay of mitoguazone dihydrochloride.

High-performance liquid chromatographic method for guanylhydrazone compounds.

A high-performance liquid chromatographic method has been developed for a series of aromatic guanylhydrazones that have demonstrated therapeutic potential as anti-inflammatory agents. The compounds were separated using octadecyl or diisopropyloctyl reversed-phase columns, with an acetonitrile gradient in water containing heptane sulfonate, tetramethylammonium chloride, and phosphoric acid. The method was used to reliably quantify levels of analyte as low as 785 ng/ml, and the detector response was linear to at least 50 micrograms/ml using a 100 microliters injection volume. The assay system was used to determine the basic pharmacokinetics of a lead compound, CNI-1493, from serum concentrations following a single intravenous injection in rats.

A novel anticancer treatment for xenoplanted human gastric cancer using polyamine antimetabolites in a low polyamine diet.

The aim of the present study was to evaluate a new anticancer treatment for gastrointestinal cancer, using a combination of polyamine antimetabolites, an anticancer agent and a low-polyamine state. Two polyamine antimetabolites, given as either 40 mg/kg of methylglyoxal-bis-guanylhydrazone (MGBG) or ethylglyoxal-bis-guanylhydrazone (EGBG) and a normal diet (ND), or 20 mg/kg of each drug and a low polyamine diet (LPD), together with 1,000 mg/kg of alphadifluoromethylornithine (DFMO) were administered ip to nude mice for six consecutive days. Mitomycin C (MMC) at 2 mg/kg was then given ip for 3 alternate days. The combination of MGBG or EGBG with DFMO plus MMC resulted in an enhanced antitumor efficacy on LPD. However, the combination which included EGBG was much more enhanced than that which included MGBG and there was no evidence of any tumor regrowth. Weight loss was minimal or nil in the mice given the combination with EGBG, but was evident in those given the combination with MGBG. These results led to the conclusion that in mice, the combined therapy of EGBG with DFMO plus MMC and LPD is a safe and effective regimen for the treatment of gastric cancer.

Chemical-analytical characterization of dihydroambazone.

Important properties of p-(thiosemicarbazido)diamino-methylen-hydrazino benzene (1; dihydroambazone) are described. The transformation of this compound into 1,4-benzoquinone guanylhydrazone thiosemicarbazone (2; ambazone) dependent on different aqueous buffer systems was investigated. The spectroscopic behaviour of the transformation product allows us to determine the content of the compound in aqueous solutions, whereas the determination in serum is much more difficult. Furthermore, TLC experiments and oxidation methods are described.

Alterations in bone marrow and blood mononuclear cell polyamine and methylglyoxal bis(guanylhydrazone) levels: phase I evaluation of alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) treatment of human hematological malignancies.

Nine patients with hematological malignancies were treated with difluoromethylornithine and methylglyoxal bis(guanylhydrazone). The number of circulating blast cells decreased in all of the patients treated with DFMO and MGBG for longer than 1 wk. Morphological evidence of myeloid maturation was evident in four patients with leukemia and the circulating M Protein decreased in one patient with multiple myeloma. The polyamine content of the mononuclear cells in both the peripheral blood and bone marrow was transiently increased after the initial MGBG dose. During administration of DFMO decreases were achieved in the peripheral blood mononuclear cell putrescine levels in 7 patients, spermidine levels in 5 patients, and spermine levels in 4 patients. Alterations in bone marrow mononuclear cell polyamine levels were similar to those which occurred in the peripheral cells. An average of 9 days of DFMO treatment was required to lower mononuclear cell polyamine levels. Three of the 4 evaluable patients receiving multiple MGBG doses had an increased mononuclear cell content of MGBG after DFMO pretreatment. Enhancement of cellular MGBG levels was not directly correlated to the degree of cellular polyamine depletion.

Rapid analysis for methylglyoxal bis(guanylhydrazone) by reversed-phase ion-pair liquid chromatography.

We describe a "high-performance" reversed-phase ion-pair liquid-chromatographic procedure for measuring methylglyoxal bis(guanylhydrazone) (MGBG) in plasma, urine, and bone-marrow leukocytes. Specimens of plasma and bone-marrow leukocytes are deproteinized with perchloric acid, then neutralized with KOH. Urinary MGBG is isolated by liquid-solid extraction in a C18 Sep-Pak. The chromatographic system consists of a 45 X 4.6 mm (i.d.) octadecylsilyl (C18, 5-microns particle) column and a mobile phase consisting of methanol/sodium acetate buffer (200 mmol/L, pH 4.5), 2/3, by vol. The acetate buffer also contains 20 mmol of 1-octanesulfonate and 40 mg of sodium azide per liter. The column effluent is monitored at 283 nm. At a flow rate of 3.0 mL/min, MGBG is eluted in 1.67 min. The detection limit is 20 nmol/L, and peak height varies linearly with concentration from 0.02 to 40 mumol/L. Analytical recovery exceeds 99%. Within-day CVs ranged from 0.9% to 2.9%, between-day CVs from 4.2% to 6.2%.

[HPLC studies on the distribution behavior of guanyl- and N-phenylguanylhydrazone derivatives]. / HPLC-Untersuchungen zum Verteilungsverhalten von Guanyl- und N-Phenylguanylhydrazonderivaten.

The article in hand presents first HPLC studies on the distribution behaviour of guanylic and N-phenylguanylic hydrazone derivates. The chromatographic parameters of compounds, which had been separated at NH2-columns by 0.06 mol X 1(-1) Na2HPO4 buffer/methanole (30 : 70), could be identified, and the k'/rates could be utilized for the calculation of the coefficient of distribution. As a result of performed calculations of the correlation, it could be proved that the log. P rates which had been ascertained by the HPLC method, been determined by the shaked flash method and been calculated acc. to Leo, correlate to the 1% significant level.

Effect of epidermal polyamine depletion on the accumulation of methylglyoxal bis(guanylhydrazone) in mouse skin.

A systemic or topical treatment of mice with alpha-difluoromethylornithine, and irreversible inhibitor of mammalian ornithine decarboxylase, produced a rapid depletion of epidermal putrescine and spermidine. When methylglyoxal bis(guanylhydrazone), another inhibitor of polyamine biosynthesis and a potent antiproliferative agent, was subsequently administered the epidermal concentration of the latter drug rose distinctly higher than without a prior difluoromethyl ornithine treatment. The combined use of these 2 antimetabolites of polyamines also profoundly depressed epidermal DNA synthesis, especially in UV-irradiated skin. A "priming" with difluoromethyl ornithine may therefore offer a means to enhance the epidermal accumulation of otherwise poorly absorbed methylglyoxal bis(guanylhydrazone).

Determination of methylglyoxal-bis(guanylhydrazone) in body fluids by ion-pair chromatography.

Methylglyoxal-bis(guanylhydrazone), Methyl-G, is a potent antineoplastic agent currently undergoing Phase l clinical trials. Serum, ascitic and pleural fluids, and urine are deproteinized with methanol, supernatant is evaporated, residue is redissolved in the eluent, lipids are removed with carbon tetrachloride, and an aliquot of the aqueous layer injected into the chromatograph. Ethylglyoxal-bis(guanylhydrazone) (Ethyl-G) is the internal standard. The mobile phase is a mixture of an aqueous buffer (containing 0.004 M heptane and pentane sulfonic acid, 90%:10%, buffered to pH 3.5) and methanol (68%:32%). The ion-pair complex is retained on a micro Bondapak C18 column, eluted with a flow of 2.0 mL/min. Absorbance is measured at 280 nm. Detectability: 30 ng/mL (0.11 micro M) in serum, ascitic and pleural fluids, 300 ng/mL (1.1 micro M) in urine. Calibration curves (peak height ratios of Methyl-G/Ethyl-G plotted against known drug concentrations) were linear in the 0.1-30 microg/mL range. Correlation coefficinets were 0.999; coefficients of variation for reproducibility were less than 5%. Residual blood levels of Methyl-G persist for several days. Methyl-G was found to pass into ascitic fluid.