Synthesis and evaluation of novel spermidine derivatives as targeted cancer chemotherapeutic agents.

The utility of the spermidine moiety as the homing device for the selective delivery of chemotherapeutic and diagnostic agents into cancer cells was explored. Two spermidine analogs containing a cytotoxic agent were synthesized, N-[3,4-bis(benzyloxy)phenethyl]-N alpha-(3-amino-propyl)-L-ornithinamide trihydrochloride, 1a and N-[4-]bis(2-chloroethyl)amino]phenethyl]-N alpha-(3-aminopropyl)-L- ornithinamide tetrahydrochloride, 1b. These compounds were prepared from the fully protected spermidine molecule with a carboxyl group side chain, 8. The ability of the polyamine cytotoxic agents to inhibit B16-BL6 melanoma cell growth in culture was examined. The effects of pretreatment with DFMO on the activity of the synthesized compounds was also studied. The IC50 values of compounds 1a and 1b were on the same order of magnitude as the control compounds, N-acetyldopamine and chlorambucil, respectively. The inhibitory activities of compounds 1a and 1b were not enhanced by pretreatment with DFMO, suggesting that depletion of intracellular polyamines did not enhance the activity of these compounds.     

多胺代谢与自噬在增龄大鼠心脏中的变化及外源性精脒对衰老心脏自噬的影响

目的探讨增龄大鼠心脏多胺代谢与自噬的变化规律,以及精脒对衰老心脏自噬的影响。方法 Westen blot法检测3、6、12、24月龄大鼠心肌多胺合成与分解代谢限速酶ODC与SSAT表达,自噬相关蛋白LC3-Ⅱ/Ⅰ与p62的表达;检测3、24月龄及给予精脒6周的24月龄大鼠心肌ATG5、ATG7、p16表达。观察心肌自噬小体形成、计算细胞横截面积及细胞凋亡率、检测活性氧(ROS)产生。结果随年龄增加,心肌ODC与LC3-Ⅱ/Ⅰ表达降低,SSAT与p62表达增加。24月龄大鼠心肌p16表达增加,细胞横截面积增大,细胞凋亡及ROS产生增多,自噬小体减少,p62表达增加,LC3-Ⅱ/Ⅰ、ATG5和ATG7表达降低。精脒干预后能明显抑制衰老诱导的以上指标的改变。结论随着年龄增加,大鼠心肌多胺合成代谢下调,分解代谢上调,细胞自噬能力下降。外源性精脒可能通过诱导心肌细胞自噬延缓大鼠心脏老化。     

1,5-(Diethylamino)piperidine, a novel spermidine analogue that more specifically activates the N-methyl-D-aspartate receptor-associated polyamine site.

We have investigated the action of 1,5-(diethylamino)piperidine (DEAP), a novel spermidine analogue that activates the polyamine site associated with the N-methyl-D-aspartate receptor. DEAP increased [3H]dizocilpine ([3H]MK801) binding to rat brain membranes with a potency similar to that of spermine and spermidine, but with a somewhat greater efficacy. Unlike other polyamines, however, DEAP did not exhibit low affinity inhibition of [3H] dizocilpine binding, suggesting that it binds more selectively to the polyamine site. DEAP increased the equilibrium affinity of [3H]dizocilpine. The increase in affinity was due to slowed dissociation, as well as a small increase in the association rate of [3H]dizocilpine. All of these effects of DEAP could be reversed by arcaine. These data illustrate the utility of DEAP as a novel polyamine agonist at the N-methyl-D-aspartate receptor complex. However, these data fail to support the hypothesis that polyamines activate the N-methyl-D-aspartate receptor by a mechanism similar to that of glutamate and glycine.     

A novel way to investigate the effects of plasma exchange on antibiotic levels: use of microdialysis

Plasma exchange (PE) is a treatment modality frequently used for many autoimmune diseases and may cause extracorporeal elimination of antibiotics. No data currently exist on antibiotic concentrations in extracellular fluid during PE. The aim of this study is to describe the effect of PE on the serum and subcutaneous tissue pharmacokinetics of piperacillin administered as a continuous infusion in a critically ill 17-year-old patient with Guillain-Barré syndrome and ventilator-associated pneumonia on Days 1 and 4 of antibiotic therapy. The effect of PE on piperacillin concentrations appears to be small. On Day 1, an estimated 7% of total piperacillin eliminated during PE was attributable to PE. On Day 4 this was estimated to be 11%. Using the in vivo sampling technique microdialysis, we have been able to show that a small redistribution of piperacillin from tissue to serum occurs in response to the reducing serum concentrations caused by PE. In critically ill patients, we believe that administration of a beta-lactam antibiotic by continuous infusion should be considered to maintain serum and tissue concentrations of these time-dependent antibiotics.     

Structure-function correlations of polyamine analog-induced increases in spermidine/spermine acetyltransferase activity

The cytosolic enzyme, spermidine/spermine acetyltransferase (SSAT), is distinguished by its role in polyamine interconversion and by its high inducibility in response to a variety of physiological and pharmacological stimuli. Among a series of fifteen polyamines and polyamine analogs, the most potent inducers of SSAT activity in cultured L1210 cells were found to be N1,N8-bis(ethyl)spermidine (BES) and N1,N12-bis(ethyl)spermine (BESm). Over a 24-hr exposure at 10 microM, enzyme activity rose 13- and 16-fold with BES and BESm, respectively, compared to 2- to 3-fold with the anticancer agent, methylglyoxal bis(guanylhydrazone). The increase in enzyme activity by BESm began rapidly and continued steadily with time so that by 48 hr it increased to about twenty times control. By inhibitor studies, the increase was found to be due to elevated protein synthesis predominantly at the level of translation and to an apparent prolongation of enzyme half-life related to enzyme stabilization. Among the analogs, the structural requirements for maximum enzyme induction were found to be critically dependent on aminopropyl moieties and on the presence, size and location of the alkyl groups. By structure-function comparisons, it was deduced that the known abilities of BES and BESm to regulate ornithine and S-adenosylmethionine decarboxylase activities or to inhibit cell growth occur independently of their effects on SSAT activity in L1210 cells.     

The use of a three compartment in vitro model to investigate the role of hepatic drug metabolism in drug-induced blood dyscrasias.

1. N-hydroxylation is thought to be an essential step in the haemotoxicity of dapsone (DDS). To investigate both metabolism-dependent and cell-selective drug toxicity in vitro we have developed a three-compartment system in which an hepatic drug metabolizing system is contained within a central compartment separated by semipermeable membranes from compartments containing mononuclear leucocytes (MNL) and red blood cells (RBC). 2. Metabolism of dapsone (100 microM) by rat liver microsomes resulted in toxicity to RBC cells (47.3 +/- 2.1% methaemoglobin), but there was no significant toxicity toward MNL (3.7 +/- 1.3% cell death) compared with control values (1.6 +/- 0.9%). However, when RBC were replaced with buffer in the third compartment there was significantly greater (P < 0.001) white cell toxicity (17.6 +/- 0.6% cell death), demonstrating the protection of MNL by RBC. Metabolism of dapsone by human liver microsomes again resulted in RBC toxicity (12.5 +/- 3.3% methaemoglobin) but no significant MNL toxicity (2.9 +/- 0.8% cell death). Replacement of RBC resulted in a significant (P < 0.001) increase in MNL toxicity (6.5 +/- 0.7% cell death). Addition of synthetic dapsone hydroxylamine (30 microM) in the absence of a metabolizing system and with no RBC in the third compartment resulted in significant (P < 0.001) toxicity toward MNL (43.36 +/- 5.82% cell death) compared with control (1.8 +/- 1.1%). The presence of RBC in the third compartment resulted in a significant (P < 0.001) decrease in MNL toxicity (17.6 +/- 2.2% cell death), with 40.1 +/- 3.7% methaemoglobin in the RBC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Small interfering RNA suppression of polyamine analog-induced spermidine/spermine n1-acetyltransferase

N1,N11-diethylnorspermine (DENSPM) is a polyamine analog that down-regulates polyamine biosynthesis and potently upregulates the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT). In certain cells, such as SKMEL-28 human melanoma cells, induction of SSAT is associated with rapid apoptosis. In this study, we used small interfering RNA (siRNA) to examine the role of SSAT induction in mediating polyamine pool depletion and apoptosis. siRNA duplexes were designed to target three independent sites in the SSAT mRNA coding region (siSSAT). When transfected under nontoxic conditions, two of the duplexes selectively reduced basal SSAT mRNA in HEK-293 cells by >80% and prevented DENSPM-induced SSAT mRNA by 95% in SK-MEL-28 cells. Treatment of SK-MEL-28 cells with 10 muM DENSPM in the presence of 83 nM siSSAT selectively prevented the 1400-fold induction of SSAT activity by approximately 90% and, in turn, prevented analog depletion of spermine (Spm) pools by approximately 35%. siSSAT also prevented DENSPM-induced cytochrome c release and caspase-3 cleavage at 36 h and apoptosis at 48 h as measured by annexin V staining. Overall, the data directly link analog induction of SSAT to Spm pool depletion and to caspase-dependent apoptosis in DENSPM-treated SK-MEL-28 cells. This represents the first use of siRNA technology directed toward a polyamine gene and the first unequivocal demonstration that SSAT induction initiates events leading to polyamine analog-induced apoptosis.     

The use of automated sequencing techniques to investigate the sequence selectivity of DNA-damaging agents

In this review, the use of automated DNA sequencing techniques to determine the sequence specificity of compounds that interact with DNA is discussed. The sequence specificity of a DNA-damaging agent is an essential element in determining the cellular mechanism of action of a drug. A number of DNA-damaging compounds are mutagenic, carcinogenic, as well as being widely used as cancer chemotherapeutic agents. The distribution of lesions in a sequence of DNA can give vital clues in the determination of the precise mechanism of interaction of the agent with DNA. The DNA sequence specificity of a number of DNA-damaging agents has been delineated using automated DNA sequencing technology, and these studies are discussed in this review. The current state-of-the-art methodology involves capillary electrophoresis with laser-induced fluorescence detection usually on an Applied Biosystems ABI 3730 capillary sequencer. This current technique has higher resolution, greater sensitivity, higher precision, more rapid separation times, is safer and easier to perform than previous methods. The two main methods to determine the DNA sequence selectivity of compounds that interact with DNA are described: end labelling and the polymerase stop assay. The interaction of the antitumour drug, bleomycin, with DNA is utilized to illustrate the recent technological advances.     

Transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N1-acetyltransferase show enhanced sensitivity to the polyamine analog, N1, N11-diethylnorspermine

We have recently generated transgenic mice in which polyamine catabolism has been activated by overexpressing the rate-limiting enzyme of polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT). These animals have now been tested for their sensitivity to the polyamine analog N1,N11-diethylnorspermine (DENSPM), which is currently undergoing Phase I clinical trial. The analog is known for its ability to potently induce SSAT. Treatment for 4 days with a daily dose (125 mg/kg) of analog caused profound changes in polyamine metabolism in the transgenic animals. Liver SSAT activity was increased by approximately 800-fold while hepatic mRNA increased only 4-fold. Putrescine pools increased while spermidine and spermine pools nearly disappeared, resulting in a compensatory increase in ornithine decarboxylase activity. Similar but less profound changes were also seen in other tissues (spleen, intestine, and skin). This treatment also resulted in a 50% mortality in the transgenic animals, with no apparent histopathological changes in major organs. Nontransgenic animals exhibited no toxicity, and tissue SSAT activity was unchanged or only moderately increased. Polyamine pools were only slightly altered. Greater analog toxicity in transgenic animals may be attributable to higher tissue levels of DENSPM facilitated by SSAT-mediated decreases in spermidine and spermine. To further confirm the enhanced sensitivity of the transgenic animals to the analog, groups of nontransgenic and transgenic animals were subjected to daily injections with DENSPM. On average, transgenic mice died approximately 3 days earlier than their nontransgenic litter-mates. The findings indicate a contributing role for SSAT in whole animal toxicity by SSAT-inducing polyamine analogs.     

Characterization of human nasal primary culture systems to investigate peptide metabolism

The objectives of this study were to validate and compare the suitability of different primary cell culture systems as models to investigate peptide enzymatic stability following nasal administration. The degradation kinetics of a model peptide, leucine enkephalin (Tyr-Gly-Gly-Phe-Leu, Leu-Enk), was determined in four nasal cell culture systems: immersion, air-liquid interface, sequential monolayer-suspension, floating collagen. The influence of enzyme inhibitors (bestatin, puromycin) and Leu-Enk metabolite analogs (Tyr-Gly, Phe-Leu, Tyr-Gly-Gly, Gly-Phe-Leu) on the Leu-Enk degradation profile was also investigated. The disappearance of Leu-Enk in all the cell culture systems followed first order kinetics. The specific activity in the cell culture systems followed the rank: sequential monolayer-suspension (32.60 microM min(-1) mg(-1)) >air-liquid interface (15.19 microM min(-1) mg(-1)) >immersion (11.49 microM min(-1) mg(-1)) >floating collagen (4.57 microM min(-1) mg(-1)). At equimolar concentration, bestatin had a higher inhibitory effect than puromycin. The rate of hydrolysis of Leu-Enk was reduced significantly by co-incubation with Leu-Enk metabolite analogs. This study showed that immersion, sequential monolayer-suspension and air-liquid interface culture systems may be potentially suitable for further studies on peptide enzymatic stability following nasal administration.     

The use of renal tubule fragments isolated from the rat to investigate aspects of gentamicin nephrotoxicity

A suspension of renal tubule fragments from the rat was prepared by a method involving collagenase digestion of the excised renal cortex and dispersion of the digest by passage through a nylon mesh. Through the use of scanning electronmicroscopy it was confirmed that the tubular lumena were patent, thus ensuring access of medium to both the luminal and the contraluminal membranes of the tubular cells. The viability of the tubule fragments was ascertained by measuring the rate of formation of glucose from pyruvate as substrate and the uptake of [¹⁴C] L-lysine against a concentration gradient. The uptake of L-lysine was unimpaired in the presence of gentamicin (10⁻³ M), suggesting that there is no competition between this basic amino acid and the polycationic aminoglycoside for transport into tubular cells. The uptake of [³H] gentamicin was studied and found to be reduced in the presence of 2,4-dinitrophenol, potassium cyanide, and ouabain. The reduced uptake in the presence of ouabain was interpreted to mean that a component of gentamicin uptake, which occurs at the luminal membrane, may be driven by the Na⁺ gradient created by Na⁺-K⁺ATPase activity at the contraluminal membrane. This renal tubule preparation offers advantages over the kidney-slice technique for studies into the mechanisms of aminoglycoside nephrotoxicity.     

The endocannabinoid system and the regulation of the metabolism

The endocannabinoid system modulates many physiological functions by acting on receptors CB1 and CB2. The endocannabinoids are produced only when and where they are needed. They act locally and are immediately metabolised after their action. Overactivation of the endocannabinoid system is observed in obesity, with stimulation of the appetite in the hypothalamus and fat accumulation in the adipocytes with increase of insulin resistance and decrease of adiponectin. Nicotine use overactivates also the endocannabinoid system. CB1 blockade by a specific inhibitor (rimonabant) decreases food intake and weight in animal studies and increases adiponectin and insulin sensitivity. Moreover, rimonabant decreases tobacco dependence. Clinical studies with rimonabant are encouraging.     

Data on the metabolism of benzylisoquinoline derivatives. The metabolism of drotaverin.

Studying the metabolism of drotaverin (No-Spa?), we have elaborated a method for the isolation, purification and separation of metabolites and of drotaverin excreted in unchanged form in the urine and feces. The structure of the chief metabolites was cleared by t.l.c., polarography, UV spectrophotometry, and mass spectrometry. It was stated that beside drotaverin excreted in unchanged form, the biotransformation of the molecule results in the formation of oxidation and desalkylation products.     

The polyamine stress response: tissue-, endocrine-, and developmental-dependent regulation

Transient alterations in polyamine (PA) metabolism, termed the polyamine stress response (PSR), constitute a common cellular response to stressful stimuli. In contrast to the adult brain and liver, the PSR in the adrenal gland and thymus is characterized by a reduction in PA metabolism. The brain PSR undergoes an early postnatal period of non-responsiveness. The aim of the present study was twofold: i) to determine whether the PSR in the liver, thymus, and adrenal gland is developmentally regulated as that in the brain and ii) to establish whether neuronal and hormonal signals can activate the PSR independently. Ornithine decarboxylase (ODC) activity and tissue PA concentrations served as markers of the PSR. Changes were measured in male Wistar rats during postnatal development and at 2 weeks after adrenalectomy in adults. Unlike the brain, the direction of the PSR in peripheral organs did not undergo developmental changes. After adrenalectomy, the PSR was not activated in the thymus and liver by acute (2-hr) restraint stress, but a characteristic PSR was induced in the hippocampus. However, dexamethasone injection (3 mg/kg) did induce a characteristic PSR in all organs of adrenalectomized rats. The results justify the following conclusions: i) Unlike peripheral organs, the PSR in the brain is developmentally regulated; ii) The developmental switch to a mature PSR in the brain corresponds in time to the cessation of the "stress hypo-responsive period" in the hypothalamic-pituitary-adrenocortical (HPA) axis; iii) In the periphery, the PSR appears to be dependent principally on stress-induced activation of the HPA axis and on increased circulating glucocorticoid concentrations rather than on neuronal activation; iv) In the brain, however, the PSR can be induced independently by glucocorticoids or by direct activation of the neuronal circuitry; and v) up-regulation of the PSR, as in the brain and liver, is constructive and may be implicated in cell survival, while its down-regulation, as in the adrenal and thymus, may be implicated in cell death.