Effect of Bis(guanylhydrazones) on Growth and Polyamine Uptake in Plant Cells.

In the present work the effect of several bis(guanylhydrazones) on the growth of Helianthus tuberosus tuber explants was studied. Different aliphatic congeners of glyoxal bis(guanylhydrazone) were tested. Most of the compounds displayed an inhibitory effect on growth, and a correlation between the structure of the molecule and the inhibitory activity was observed. Experiments carried out with glyoxal bis(guanylhydrazone) and its congeners methyl-, ethylmethyl-, and methylpropylglyoxal bis(guanylhydrazones) show that as the total number of side chain carbon atoms in the molecule increases, the inhibitory potency also increases. A depletion of spermidine levels was also found in the explants treated with ethylmethylglyoxal bis(guanylhydrazone), which turned out to be one of the most potent growth inhibitors. The addition of spermidine caused a significant reversion of the antiproliferative action of glyoxal bis(guanylhydrazone). The effect of these compounds on spermidine uptake in protoplasts isolated from carrot phloem parenchyma was also investigated. Only a slight competition was found when antagonists were present at concentrations 20 times higher than the polyamine, thus suggesting that bis(guanylhydrazones) do not share, at least at low concentrations, the polyamine transport system in plant cells.Key Words. Bis(guanylhydrazones)-Carrot protoplasts-Growth-Helianthus tuberosus-Polyamines-Uptakehttp://link.springer-ny.com/link/service/journals/00344/bibs/18n1p39.html

Comparison of pharmacokinetics of clodronate after single and repeated doses.

OBJECTIVE: Pharmacokinetics of orally given clodronate disodium, a drug for the treatment of hypercalcemia and bone resorption, were studied after a single dose of 400, 800 and 1600 mg given randomly to 11 healthy volunteers in a crossover manner, in 7-14 hospitalized cancer patients given 400, 800 and 1600 mg twice daily, each dosage for one week, and during the customary therapy in 15 additional cancer patients treated in hospital with 400 mg thrice daily for > or = 2 weeks. METHODS: Clodronate concentrations in serum and urine were measured by capillary gaschromatography with mass-selective detection. Pharmacokinetic parameters were calculated with a three-compartmental model. RESULTS: After a single oral dose to healthy volunteers the absolute clodronate concentrations increased almost dose-dependently. The mean cumulative excretion in urine was 1.72-2.77% of the dose, an interindividual range being from 0.92% to 5.52%. With 800 and 1600 mg twice daily for one week to cancer patients the serum drug concentrations increased almost progressively with increasing the dose. In cancer patients serum drug concentrations were clearly higher and renal drug clearances (mean 25-62 ml/min) lower than in healthy volunteers (mean 123-149 ml/min). The mean urinary excretions were 2.24-3.14% of the dose and interindividual ranges from 0.18% to 19.0%. During the routine cancer therapy with 400 mg thrice daily, the clodronate excretions in urine on two successive days were on an average 3.26% (range 0.0-10.5%). CONCLUSIONS: Absolute concentrations in serum and excretions in urine of orally given clodronate increase dose-dependently, but during the maintenance therapy in hospitalized cancer patients the renal drug clearances seem to be lower than in healthy volunteers. This and the large interindividual variation in kinetics propose therapeutic monitoring of clodronate for optimizing the oral dose of the drug.

Determination of the antileukemic drug mitoguazone and seven other closely related bis(amidinohydrazones) in human blood serum by high-performance liquid chromatography.

A reversed-phase (C18) HPLC method with diode-array detection was developed for the separation and determination of methylglyoxal bis(amidinohydrazone) (mitoguazone) and seven closely related aliphatic analogs thereof, namely the bis(amidinohydrazones) of glyoxal, dimethylglyoxal, ethylmethylglyoxal, methylpropylglyoxal, butylmethylglyoxal, diethylglyoxal and dipropylglyoxal. The mobile phase consisted of a non-linear binary gradient of methanol and 0.03 M aqueous sodium acetate buffer (pH 4.3). Good separation of the eight congeners was achieved. On increasing the methanol content of the eluent, the bis(amidinohydrazones) eluted in the order of increasing number of carbon atoms in the side-chains. The method was also applied to the quantitative analysis of the compounds in aqueous solution and, combined with ultrafiltration, for the separation of the eight congeners in spiked human blood serum. A separate simplified method for the quantitative determination of each of the compounds in spiked human blood serum samples was also developed. The methods developed made for the first time possible the simultaneous HPLC analysis of more than one bis(amidinohydrazones). The results obtained indicate that the bis(amidinohydrazones) studied obviously have a distinct tendency to form ion associates with acetate ions and probably also other carboxylate ions in aqueous solution. This aspect may be of biochemical significance, especially concerning the intracellular binding of the compounds. Each one of the compounds studied invariably gave rise to one peak only, this result supporting the theory that the conventional synthesis of each of the compounds gives rise to one geometrical isomer only. This result is completely in agreement with the results of previous proton and carbon NMR spectroscopic as well as X-ray diffraction studies.

Biochemical and chemical characterization of phenylglyoxal bis(guanylhydrazone), an aromatic analogue of mitoguazone.

Since little has been known about the properties of aromatic analogues of the antineoplastic agent methylglyoxal bis(guanylhydrazone) (MGBG), an investigation was performed on phenylglyoxal bis(guanylhydrazone) (PhGBG). PhGBG competitively inhibited yeast adenosylmethionine decarboxylase (AdoMetDC) with a Ki of 65 microM. As compared to MGBG (Ki 0.23 microM), PhGBG is a much weaker inhibitor, being even weaker than the unsubstituted congener glyoxal bis(guanylhydrazone) (GBG, Ki 18 microM). PhGBG inhibited porcine kidney diamine oxidase (DAO) non-competitively, being a more potent inhibitor (Ki 0.12 microM) than GBG (Ki 0.17 microM) or MGBG (Ki 0.33 microM). Thus, PhGBG has an unfavourably high ratio of Ki(AdoMetDC)/Ki(DAO) for potential use for selectively inhibiting polyamine biosynthesis. This does not exclude the possibility that PhGBG or other aromatic congeners might have therapeutic value since the corresponding ratio of the antileukaemic congeners GBG and MGBG is also high as compared to many aliphatic non-antileukaemic analogues. The pKa1 and pKa2 values of PhGBG dication were found to be 6.39 +/- 0.02 and 8.64 +/- 0.02 respectively, their difference being distinctly larger than in the case of GBG or its C-alkylated analogues. This may result from decreased stability of the dication form, caused by the resonance effect or possibly by the inductive effect of the phenyl group. The species distribution of PhGBG (proportion of free base 5.5%, predominant species the monocation) at 37 degrees C resembles that of GBG and MGBG but is clearly different from that of non-antileukaemic C-alkylated analogues. These similarities suggest that PhGBG and its derivatives may be worth antitumour screening. Depending on the conditions used in the crystallization, three different types of crystals of PhGBG sulphate were obtained. Crystallography indicated that, in two of the types, the crystal consisted exclusively of the anti-anti isomer, i.e. the same isomer as has been observed in the case of GBG and its C-alkylated congeners. One crystal type, however, consisted of a different geometrical isomer (anti-syn), suggesting that PhGBG may isomerize more easily than its aliphatic analogues. Previous concepts on the isomerism of GBG and C-alkylated bis(guanylhydrazones) thus cannot be generalized to aromatic congeners. A theory based on resonance, inductive and hyperconjugative effects and electron transfers is presented that is capable of explaining the formation of the two geometrical isomers of PhGBG that were experimentally observed. A similar theory, based on hyperconjugation of C-F bonds, is presented that is capable of explaining the previous finding of the formation of the anti-syn isomer of trifluoromethylglyoxal bis(guanylhydrazone) (CF3GBG). Like that of CF3GBG, the anti-syn isomer of the PhGBG dication is stabilized by an internal hydrogen bond. The lack of structural rigidity may affect the biological properties of PhGBG, e.g. its ability to inhibit AdoMetDC.

Determination of bis(amidinohydrazones) by micellar electrokinetic capillary chromatography.

A micellar electrokinetic capillary chromatography method was developed for the separation and determination of aliphatic congeners of bis(amidinohydrazones) in standard solution. Eight bis(amidinohydrazones) could be determined in less than 15 min at an applied voltage of 22 kV, using 0.05 M sodium phosphate as buffer (pH 7.0) together with 1 mM N-cetyl-N,N,N-trimethylammonium bromide. Hydrostatic sample injection was employed. The method exhibited good repeatability and a linear range of 2.5-100 micrograms ml-1. A detection limit of 1 micrograms ml-1 was achieved. The method also allows the determination of bis(amidinohydrazones) in human serum samples.

Inhibition of human and rabbit platelet aggregation by chlorophenoxyacid herbicides.

Inhibition of human platelet aggregation by eight chlorophenoxyacid herbicides was studied in vitro. Thrombocyte aggregation in the platelet-rich plasma was induced by 1.0-32.0 microM adenosine diphosphate (ADP), 0.32-32.0 microM adrenaline or 7.5-30.0 micrograms/ml collagen with and without chlorophenoxyacid (0.05-2.0 mg/ml). Platelet aggregation by each inducer was inhibited dose dependently by all the eight chlorophenoxyacids at concentrations between 0.1 and 2.0 mg/ml. Increasing the concentrations of ADP and collagen but not of adrenaline inhibited the antiaggregatory action of chlorophenoxy-acids. No essential differences in inhibitory effect were found between different chlorophenoxyacids varying in respect of their ring substituents and the length of the carboxylic side chain. In the platelet-rich plasma prepared from rabbits 2.5 h after subcutaneous injection of 2.4-dichlorophenoxyacetic acid or 4-chloro-2-methylphenoxyacetic acid (100-150 mg/kg), platelet aggregation by ADP was inhibited 20-30%, compared to plasma taken from the rabbits before the chlorophenoxyacid treatment. The inhibition had disappeared by 20-23 h after administration. The results indicate that chlorophenoxyacid herbicides inhibit human platelet aggregation. Furthermore, the inhibition is probably involved in haemorrhages known to occur in various tissues of animals intoxicated by chlorophenoxyacid herbicides.

Adenosylmethionine decarboxylase inhibitors--lack of activity against cytopathic effects of HIV.

Many bis(amidinohydrazones) are potent inhibitors of adenosylmethionine decarboxylase (AdoMetDC), a key enzyme of polyamine biosynthesis, and some of them are also known to be powerful antiviral agents. Therefore, seven bis(amidinohydrazones), including the two most potent inhibitors of eukaryotic AdoMetDC so far reported, were screened for antiviral activity against the human immunodeficiency virus (HIV). The screening was performed by incubating susceptible human leukemia cells in microculture plates in the presence or absence of test compounds for 7 days and by determining the number of viable cells at the end of the test. None of the compounds screened, however, displayed any detectable antiviral activity (i.e. none of them increased the viability of virus-infected cells) in these tests whose aim was to reveal potential activity against the cytopathic effects of HIV. This result suggests that inhibitors of AdoMetDC, at least when used alone, are devoid of value for the prevention of the cytopathic effects of HIV. However, the possibility cannot be totally excluded that some of them might decrease the amount of infectious progeny viruses formed, just as methylglyoxal bis(amidinohydrazone) is known to do in the case of vaccinia virus.

Distribution of three common chlorophenoxyacetic acid herbicides into the rat brain.

The distribution of three common 14C-labelled chlorophenoxyacetic acid herbicides (2,4-dichlorophenoxyacetic acid or 2,4-D, 2-methyl-4-chlorophenoxyacetic acid or MCPA, 2,4,5-trichlorophenoxyacetic acid or 2,4,5-T) into the different brain areas was studied in rats pretreated with toxic doses of the herbicides (238-475 mg/kg). Also, their binding to proteins in rat plasma was determined in vitro by increasing the concentrations of chlorophenoxyacetic acids in the incubate from 0 to 1 mg/ml. Both 2,4-D and MCPA pretreatments increased brain concentrations of 14C-labelled herbicides more markedly than 2,4,5-T pretreatments did. No essential differences were found in the distribution between the different brain areas. Protein-unbound fractions of 2,4-D and MCPA in the plasma were clearly higher than those of 2,4,5-T but the highest herbicide concentration increased the protein-unbound fraction of 2,4,5-T more (7-13-fold) than of 2,4-D and MCPA (5-fold). The results suggest that the greater increase in the penetration into the brain of 2,4-D and MCPA than of 2,4,5-T during their intoxication is due to some factors other than the changes in their binding to plasma proteins and mere enhanced diffusion through the blood-brain barrier.

Increase in the acute toxicity and brain concentrations of chlorophenoxyacetic acids by probenecid in rats.

1. Probenecid increased the acute toxicity of chlorophenoxyacetic acids (2,4-D, 2,4,5-T and MCPA) in rats. 2. Probenecid increased the brain to plasma ratios of all the three 14C-labelled chlorophenoxyacetic acids. The increase was due only partly to the displacement of chlorophenoxyacids from their binding sites in rat plasma proteins by probenecid. 3. Probenecid did not change significantly the intracerebral distribution pattern of 14C-labelled chlorophenoxyacetic acids.

Effect of central muscle relaxants on single-dose pharmacokinetics of peroral paracetamol in man.

Paracetamol (acetaminophen) at a single, 160-450 mg dose was given perorally in combination with central muscle relaxants (CMRs) carisoprodol (200 mg), chlormezanone (100 mg) or orphenadrine (35 mg) in a double-blind, randomized, cross-over study in 10 healthy volunteers. The pharmacokinetic parameters of paracetamol remained unaltered in the presence of the CMRs as compared with those observed after paracetamol without additives, in spite of nearly twenty-fold differences in the dissolution rate between the products. Paracetamol is absorbed mostly in the duodenum, and therefore there is enough time for even the slowly dissolving tablets to release the active principle before the gastric contents are transferred to the area of paracetamol absorption. Some CMRs are anticholinergic compounds and may affect intestinal motility. Our results show, however, that CMRs do not significantly alter the pharmacokinetics of paracetamol, and presumably the antipyretic or analgetic efficacy of paracetamol is not impaired when combination formulations of paracetamol and CMRs are used.

Remarkable differences between the species distributions of various bis(guanylhydrazones) at physiological conditions, and their possible involvement in the strict structural requirements for antileukemic activity.

The first systematic study on the acid-base properties of the antileukemic agents glyoxal bis(guanylhydrazone) (GBG) and methylglyoxal bis(guanylhydrazone) (MGBG) and of their non-antileukemic monoalkyl- and dialkylglyoxal analogs is reported. At physiological conditions (pH 7.4, 37 degrees C), the species distribution of GBG and MGBG differs remarkably from that of their inactive congeners, a noteworthy proportion of GBG (10.2%) and MGBG (4.0%) existing in the form of the free base while the corresponding proportion of their non-antiproliferative analogs is only 0.5% or less. Ethylglyoxal bis(guanylhydrazone) (EGBG), which has antiproliferative properties in vitro but is devoid of antileukemic activity in vivo, is intermediate between the two groups, 2.6% of it existing in the free base form. In contrast to what has been generally assumed, at physiological conditions, the predominant species of GBG, MGBG, and EGBG is the monocation form and not the dication. Considerable proportions of other congeners also exist in the monocation form. At slightly higher pH values that are of interest because of the known antimitochondrial effects of GBG and MGBG (and, in high concentrations, EGBG), the species distribution of GBG and MGBG differs even more remarkably from that of the dialkylglyoxal analogs. Thus, at pH 8.0 and 37 degrees C, as much as 36% of GBG and 19% of MGBG exist in the free base form, the corresponding proportion of EGBG being 14% and that of the other congeners studies only ca. 3-4%. On the basis of the results, it appears possible that the unusually strict structure-activity relationships of this class of antineoplastic agents may be based on the remarkable differences between the species distributions of the various congeners. The hypothesis is presented that the actual antiproliferative and antimitochondrial species of the compounds is the free base form. A compilation of pKa1 and pKa2 values, measured by potentiometric methods in 0.1 M NaCl (aq) at 25 degrees C and 37 degrees C, is given for six bis(guanylhydrazones). The species distribution curves of the compounds (at 37 degrees C) are given for the pH range 6-10.

The uptake of clodronate (dichloromethylene bisphosphonate) by macrophages in vivo and in vitro.

Clodronate (dichloromethylene bisphosphonate) accumualtes extensively in the bone by binding to apatite crystals. We found recently that the drug also accumulates in the spleen and, to a lesser extent, in the liver of mice and rats. In the present study, the role of macrophages in soft tissue accumulation was studied in mice by autoradiography and macrophage-depleting techniques, and also by isolated rat peritoneal macrophages. The localization of [14C]clodronate in mouse spleen showed that the drug concentrates in the marginal zone between the white and red pulp, which is known to be rich in macrophages. Pretreatment of mice with pure clodronate did not change the accumulation of [14C]clodronate in the spleen. However, when splenic and hepatic macrophages were eliminated by the liposome-encapsulated clodronate, only a weak [14C]clodronate accumulation occurred in these organs. Isolated macrophages did not take up free [14C]clodronate, but the addition of ferrous iron to the incubate resulted in the uptake of 14C activity by macrophages. They were probably stimulated by the insoluble clodronate-iron complex. The results suggest that 1) macrophages are involved in the accumulation of clodronate in the spleen and liver, and 2) combination of clodronate with extracellular iron is a prerequisite for the activation of macrophages to take up the drug complex. Since the spleen and, to a lesser extent, the liver are rich in iron released from destroyed red cells, accumulation of clodronate takes place in these organs.

Effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on biogenic amines and their acidic metabolites in brain and cerebrospinal fluid of rats.

Effects of single subcutaneous doses of sodium 2,4-dichlorophenoxyacetate (2,4-D-Na) on biogenic amines and their acidic metabolites in rat brain and cerebrospinal fluid (CSF) were analyzed by high pressure liquid chromatography. After 200 mg/kg 2,4-D-Na, the cerebral concentration of 5-hydroxytryptamine (5-HT) was increased slightly and that of 5-hydroxyindoleacetic acid (5-HIAA) roughly 3-fold between 1 and 8 h after the administration. There was also a tendency towards slightly lowered dopamine (DA) levels. No statistically significant changes in brain concentrations of noradrenaline (NA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) or tryptophan (TRY) were found. At the same time, however, the maximal increase in DOPAC, HVA and 5-HIAA concentrations in the CSF was 2.3-5.8-fold. The dependency of biogenic amines and metabolites on 2,4-D-Na dose was studied by injecting s.c. 0, 10, 30 and 100 mg/kg and sacrificing the rats at 2 h. In the brain, there was a dose-dependent increase in concentrations of 5-HIAA (at the two highest doses) and HVA (at the highest dose) while in the CSF those of all three acidic metabolites increased at the two highest doses. The 10 mg/kg dose had no effect. The results agree with the hypothesis that 2,4-D inhibits the organic acid transport out of the brain, which should then result in increased cerebral levels of acidic metabolites of biogenic amines, but it may also have effects on the activity of serotoninergic and dopaminergic neurones.

Biochemical and chemical characterization of trifluoromethylglyoxal bis(guanylhydrazone), a close analog of the antileukemic drug mitoguazone.

In order to study the structure-activity relationships of bis(guanylhydrazone) type polyamine antimetabolites, trifluoromethylglyoxal bis(guanylhydrazone) (CF3-GBG), a close analog of the antileukemic drug methylglyoxal bis(guanylhydrazone) (mitoguazone, MGBG) was synthesized according to a novel modification of previous methods, yielding single crystals. Single-crystal X-ray crystallography revealed the presence of an isomer different from the one detected in the case of MGBG and all other bis(guanylhydrazones) so far studied. In contrast to MGBG, CF3-GBG was shown to be a very weak inhibitor of yeast adenosylmethionine decarboxylase, being thus devoid of value as a polyamine antimetabolite. In addition, the compound did not have antiproliferative activity against mouse L1210 leukemia cells in vitro. As long as analogous isomers of the two compounds are not available, no conclusions can be drawn about the reasons lying behind the drastical differences between their biological properties.

Diethylglyoxal bis(guanylhydrazone): a novel highly potent inhibitor of S-adenosylmethionine decarboxylase with promising properties for potential chemotherapeutic use.

Diethylglyoxal bis(guanylhydrazone) (DEGBG), a novel analog of the antileukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) was synthesized. It was found to be the most powerful inhibitor of yeast S-adenosylmethionine decarboxylase (AdoMetDC) so far studied (Ki approx. 9 nM). This property, together with the finding that the compound is a weaker inhibitor of intestinal diamine oxidase than are MGBG and its glyoxal, ethylglyoxal and ethylmethylglyoxal analogs, makes the compound a promising candidate as a polyamine antimetabolite for chemotherapy studies. DEGBG was also found to potentiate the antiproliferative effect of the ornithine decarboxylase inhibitor alpha-difluoromethyl ornithine against mouse L1210 leukemia cells in vitro. DEGBG increased several-fold the intracellular putrescine concentration of cultured L1210 cells, just as MGBG and its ethylglyoxal analog are known to do. The results strongly suggest that DEGBG is worth further studies. Combined with previous studies, they also made possible the construction of some empirical rules concerning the structure-activity relationships of bis(guanylhydrazone) type inhibitors of AdoMetDC. The identity of DEGBG was confirmed by a single-crystal X-ray analysis and by 1H- and 13C-NMR spectroscopy. It consisted of the same isomer as MGBG and several of its analogs are known to consist of.

Comparative study on cerebrovascular injuries by three chlorophenoxyacetic acids (2,4-D, 2,4,5-T and MCPA).

At toxic doses in rats, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) caused injuries in blood vessels in the brain. 2. 2,4-D caused extravasations of the circulating Evans blue-albumin complex also in the spinal cord. 3. By contrast, potency in damaging cerebral vessels was almost non-existent with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). 4. None of the three chlorophenoxyacetic acids caused such cerebrovascular injuries in mice, guinea pigs, Syrian hamsters, rabbits and chickens.

An antiproliferative salicylaldoximato type copper(II) chelate as an enhancer of the cytotoxicity of murine spleen cells to tumor cells in vitro.

The antiproliferative metal chelate bis(2,3,4-trihydroxybenzaldoxyimato)copper(II) (CuTRI2) was shown to distinctly enhance the cytotoxicity of the spleen effector cells of CBA/CA mice to YAC-1 tumor cells in vitro as measured using a 4-h 51Cr release test. The compound was either added directly to the assay system or the effectors were incubated in the presence of it for 2 h and washed before assay. The concentrations tested were 10, 20 and 40 mg/ml, the lowest concentration leading invariably to the strongest enhancement of cytotoxicity. The effector cells involved were very probably natural killer cells. The highest concentration had, if anything, a negative effect on the release of 51Cr from the target cells. In the same concentrations, CuTRI2 did not enhance the cytotoxicity of the spleen effector cells to K-562 tumor cells. Preliminary studies on the related chelate trans-bis(2,4-dihydroxybenzaldoximato)copper(II) are also reported. This paper is apparently the first report of a copper chelate that enhances the cytotoxicity of any sort of effector cells to tumor cells.

Distribution of [14C]clodronate (dichloromethylene bisphosphonate) disodium in mice.

The distribution of 14C-labeled clodronate (dichloromethylene bisphosphonate), a new bisphosphonate for the treatment of osteolytic bone metastases and hypercalcemia, was studied in mice by whole-body autoradiography and by measuring the 14C activities in various tissues [14C]Clodronate was administered into the tail vein, and its distribution was followed from 5 min to 90 days after the injection. The drug disappeared promptly from the plasma and accumulated intensively in the bone and moderately in the spleen. In both tissues, relatively high radioactivities were measured as late as 90 days after the [14C]clodronate administration. Small amounts of 14C activity were also detected in the liver for 90 days. The results agree well with the previous observations that bisphosphonates deposit rapidly in the bone. Our findings indicate further that clodronate accumulates in the bone and the spleen for several months.