Potent antitumor activity and improved pharmacological profile of ST1481, a novel 7-substituted camptothecin.

Relevant drawbacks of the molecular structure and mechanism of the action of camptothecins are the instability of the E ring lactone and the reversibility of drug-target interaction. Such features are expected to limit the clinical efficacy of conventional camptothecins. In an attempt to overcome these limitations and to improve the pharmacological profile of camptothecins, a novel series of seven modified lipophilic analogues was synthesized based on the hypothesis that lipophilicity could promote a rapid cellular accumulation and stabilization of drug-target interaction. A novel analogue (ST1481) of the series, characterized by a potent antitopoisomerase and cytotoxic activity, was selected for preclinical development. A detailed preclinical study of ST1481 was performed in the H460 non-small cell lung tumor model using oral administration and various treatment schedules. Under all of the conditions, ST1481 exhibited an impressive efficacy in terms of tumor growth inhibition (tumor volume inhibition percentage > 99%), log(10) cell kill, rate of complete responses (including "cures"), and an improvement of the therapeutic index compared with topotecan (used as the reference drug). The cytotoxic potency was also reflected by the in vivo potency, because the drug activity was observed at doses as low as 0.25 mg/kg with the daily schedule. In contrast to topotecan, no cross-resistance to ST1481 was found in ovarian carcinoma cells overexpressing P-glycoprotein (A2780/DX). A similar trend in the improvement of activity was also observed in the same tumor model growing in vivo with a 100% rate of complete tumor regressions. A rapid intestinal absorption and good oral bioavailability were supported by in vivo distribution studies, because the peak values of drug accumulation were found from 1 to 2 h after administration. The relevant liver accumulation may account for a marked effect of ST1481 against liver metastases induced by the ovarian carcinoma IGROV-1. In conclusion, the results support the hypothesis that a potent lipophilic camptothecin with a proper substituent at the position 7 may have therapeutic advantages likely related to a rapid intracellular uptake and tissue distribution, stabilization of the drug-target complex, and good oral bioavailability. Overall, the results support the preclinical interest of ST1481 in terms of efficacy, potency, toxicity profile, and ability to overcome multidrug resistance.

Bovine brain ketimine reductase.

We report the purification from bovine brain of an NAD(P)H-dependent reductase which actively reduces a new class of cyclic unsaturated compounds, named ketimines. Ketimines arise from the transamination of some sulphur-containing amino acids, such as L-cystathionine, S-aminoethyl-L-cysteine and L-lanthionine. The enzyme also reduces delta 1-piperidine 2-carboxylate, the carbon analog of aminoethylcysteine ketimine. Some kinetic and molecular properties of this enzyme have been determined. Subcellular localization and regional brain distribution have also been studied. The ketimine reductase activity was found to be associated with the soluble fraction, and was located prevalently in the cerebellum and cerebral cortices. Cyclothionine and 1,4-thiomorpholine-3,5-dicarboxylic acid, the enzymatic reduction products of cystathionine ketimine and lanthionine ketimine, respectively, have been detected in bovine brain, thus suggesting a role of this enzyme in their biosynthesis.

Detection of 2H-1,4-thiazine-5,6-dihydro-3,5-dicarboxylic acid (lanthionine ketimine) in the bovine brain by a fluorometric assay.

A new sulfur imino acid, 2H-1,4-thiazine-5,6-dihydro-3,5-dicarboxylic acid (lanthionine ketimine), has been detected in the bovine brain by means of fluorometric and HPLC procedures. The fluorometric assay is based on the fluorescent property of the copper-ketimine interaction product at pH 11.5. Other ketimines do not fluoresce in these conditions. The fluorophore exhibits an excitation maximum at 353 nm and an emission at 462 nm and is stable for at least 24 h. In the test conditions the fluorescence is proportional to the ketimine concentration from 1 to 200 microM. Detection of endogenous lanthionine ketimine has been performed after a simple enrichment procedure (brain deproteinization and extraction with diethyl ether) which minimizes degradative by-reactions of the unstable ketimine. The concentration of this new sulfur imino acid in the brain ranges from 0.5 to 1 nmol/g in three different samples. Identification and quantitations were confirmed by an HPLC procedure which takes advantage of the selective absorption at 380 nm of the phenylisothiocyanate-ketimine adduct. The identification of lanthionine ketimine in nervous tissues may have important metabolic and physiological implications.

Resistance to the atypical retinoid ST1926 in SK-N-AS cells selected the subline rAS-ST with enhanced sensitivity to ATRA mediated by not conventional mechanisms: DNA damage, G2 accumulation and late telomerase inhibition.

BACKGROUND AND PURPOSE: 13-cis-Retinoic acid represents a well-established clinical strategy for the management of minimal residual disease of high risk neuroblastoma (NB) patients. However, the clinical efficacy on the overall survival of these patients remains limited, addressing the issue of better understanding the molecular mechanisms and intracellular pathways mediating Retinoic Acid (RA) clinical effects. EXPERIMENTAL APPROACH: This work investigates the mechanism underlying the sensitivity/resistance to RA in NB by taking advantage of the paired SK-N-AS/rAS-ST cells showing different responsivity to ATRA. The subline rAS-ST was selected by inducing resistance to the novel retinoid ST1926 in the NB SK-N-AS cell line. KEY RESULTS: Resistance to ST1926 was neither dependent on cellular uptake nor on multi-drug resistance phenotype. Rather, both delayed/lower DNA damage and apoptosis appeared involved in reduced sensitivity of rAS-ST cells to ST1926. This subline showed enhanced responsivity to ATRA compared to the wt counterpart, that was associated with enhanced RARα/ß expression, DNA damage, G2 accumulation, PI3K/AKT pathway inhibition, cellular differentiation and delayed telomerase inhibition, without involvement of either p27/p53 or caspase-mediated apoptosis. CONCLUSIONS AND IMPLICATIONS: The present data add important information to the understanding of RA sensitivity in NB, providing further insights towards a more efficacious clinical use of this drug.

Displacement of [(3)H]GABA binding to bovine brain receptors by sulfur-containing analogues.

The displacement of [(3)H]GABA binding to GABA receptors of bovine brain cortical membranes by some sulfur-containing compounds (homothiotaurine, thiotaurine and carboxymethylcysteamine) was investigated and their potency was compared to that of other known sulfur-containing analogues of GABA, such as homotaurine, homohypotaurine and taurine. Displacement studies showed homotaurine to be more effective as a GABA displacer than homohypotaurine and homothiotaurine (IC(50): 3.9 x 10(?8), 6.7 x 10(?7) and 6.8 x 10(?7) M, respectively). Saturation experiments showed that the effect of taurine, homothiotaurine, homotaurine and homohypotaurine was due to a loss of high-affinity GABA sites (K(d) = 10.7 nM). Homotaurine seems also to interact with low-affinity sites, decreasing the affinity constant, whereas the number of binding sites remains unchanged.

Decreased density of endothelin binding sites in adrenal glands but not in aorta, of long term infarcted rats.

Endothelins (Et-s) are biologically active peptides which play a physiological and pathological role in the cardiovascular regulation. The aim of our study was to verify, in a model of experimental long term myocardial ischemia (15 weeks) in rats, whether there was a modification in the ET binding sites of aorta and adrenal glands. Additionally, Ang II binding sites in adrenal glands were studied. The principal finding of the present study was the down-regulation of ET binding sites in adrenal glands of chronic infarcted rats, whereas no modification of binding parameters for Et-1, in thoracic aorta, nor for Ang II, in adrenal glands, were found.

Antitumor activity of the combination of synthetic retinoid ST1926 and cisplatin in ovarian carcinoma models.

BACKGROUND: The novel adamantyl retinoid ST1926 is a potent inducer of apoptosis in ovarian carcinoma cells. Since the pro-apoptotic effect is associated with activation of p53, in this study we have investigated the efficacy of combination of ST1926 with cisplatin, a DNA-damaging agent that is known to induce p53-dependent apoptosis. MATERIALS AND METHODS: The efficacy of ST1926 and its combination with cisplatin was evaluated in human ovarian carcinoma models, including resistant tumors. RESULTS: Oral treatment with ST1926 alone caused a marginal tumor growth inhibition (<50%), but the combination with cisplatin resulted in an improved efficacy, most evident in terms of tumor growth delay without a substantial increase of toxicity. The combination therapy achieved the best effects against the HOC18 ovarian carcinoma tumor, resulting in an appreciable number of animals without evidence of disease at the end of the experiment. In contrast to the marginal effect of ST1926 alone against the subcutaneous-growing tumors, loco-regional (intraperitoneal) treatment achieved a marked increase of survival of animals with ascitic IGROV-1 tumor. CONCLUSIONS: The present results document the efficacy of the combination of cisplatin with ST1926 and provide a rational basis for the design of novel, well-tolerated platinum-based treatment approaches in human ovarian carcinoma.

Characterization and subcellular localization of L-[3H] carnitine binding sites in rat brain.

In the present study, we investigated the existence of a binding site for L-carnitine in the rat brain. In crude synaptic membranes, L-[3H]carnitine bound with relatively high affinity (KD = 281 nM) and in a saturable manner to a finite number (apparent Bmax value = 7.3 pmol/mg of protein) of binding sites. Binding was reversible and dependent on protein concentration, pH, ionic strength, and temperature. Kinetic studies revealed a Koff of 0.018 min-1 and a Kon of 0.187 x 10(-3) min-1 nM-1. Binding was highest in spinal cord, followed by medulla oblongata-pons > or = corpus striatum > or = cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. L-[3H]Carnitine binding was stereoselective for the L-isomers of carnitine, propionylcarnitine, and acetylcarnitine. The most potent inhibitor of L-[3H]carnitine binding was L-carnitine followed by propionyl-L-carnitine. Acetyl-L-carnitine and isobutyryl-L-carnitine showed an affinity approximately 500-fold lower than that obtained for L-carnitine. The precursor gamma-butyrobetaine had negligible activity at 0.1 mM. L-Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glutamate, aspartate, acetycholine, dopamine, norepinephrine, epinephrine, 5-hydroxytryptamine, histamine) at a final concentration of 0.1 mM. In addition, the binding of these neuroactive compounds to their receptors was not influenced by the presence of 0.1 mM L-carnitine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac and renal endothelin-1 binding sites in streptozotocin-induced diabetic rats.

UNLABELLED: The aim of this work was to study cardiac and renal endothelin binding sites during the progression of diabetes. Male Crl:CD (BR) rats were made diabetic by injection of streptozotocin (STZ, 45 mg kg-1 i.v.). Only rats with a glycaemia of 500 mg per 100 ml or higher, were used. The hearts were taken at 2, 4 or 6 weeks and kidneys at 2 and 6 weeks, after diabetes induction, for binding studies. In the heart, the number of Et-1 binding sites was significantly increased 2 weeks after STZ-induction of diabetes (449 +/- 13 vs. 345 +/- 18 fmol (mg protein) -1, in controls; p < 0.05) without modification of KD value (104 +/- 5 vs 101 +/- 7 pM). Comparable results were obtained 4 and 6 weeks after STZ-induction. In the kidney both the parameters were unchanged at all the times tested. IN CONCLUSION: a specific increase in cardiac Et-1 binding sites, without change in affinity of the peptide, was found 2, 4 and 6 weeks after diabetes induction; while renal Et-1 binding sites were not modified.

Purification and characterization of a ketimine-reducing enzyme.

An NAD(P)H-dependent reductase able to reduce a new class of cyclic unsaturated compounds named ketimines has been detected and purified 2500-fold from pig kidney. Some molecular and kinetic properties of this enzyme have been determined. The enzymatic reduction proceeds with a classical ping-pong mechanism and some results suggest that the true substrate has the ketiminic structure and is in equilibrium with the enaminic and keto-open forms. As previously described, ketimines arise from the deamination of a number of sulfur-containing amino acids, i.e. L-cystathionine, L-lanthionine and S-aminoethyl-L-cysteine, catalyzed by a widespread mammalian transaminase. The enzymatic reduction products of ketimines have been identified as cyclothionine, 1,4-thiomorpholine 3,5-dicarboxylic acid and 1,4-thiomorpholine 3-carboxylic acid. Some of these compounds have been detected in mammals, thus suggesting a possible role of this enzyme in their biosynthesis.

Detection of cystathionine ketimine in bovine cerebellum.

A new sulfur-containing cyclic imino acid, cystathionine ketimine, has been detected in bovine cerebellum by gas chromatography, gas chromatography-mass spectrometry, and high pressure liquid chromatography procedures. Gas chromatography and gas-mass analyses are based on derivatization of endogenous cystathionine ketimine with diazomethane after a simple enrichment procedure. The high pressure liquid chromatography procedure takes advantage of the selective absorbance at 380 nm of the phenyl isothiocyanate-ketimine interaction product. The concentration of this new sulfur imino acid found in a pool of four bovine cerebella is approximately 0.5 nmol/g.

Gastrosparing effect of new antiinflammatory drug amtolmetin guacyl in the rat: involvement of nitric oxide.

The effect of the nonsteroidal antiinflammatory drug (NSAID) amtolmetin guacyl (AMG) on the gastric mucosa was studied in the rat by means of histological and functional techniques. AMG administered at 50-300 mg/kg intragastrically was virtually devoid of gastrolesive properties after either acute or repeated treatment. By contrast, its metabolite, tolmetin (TOL, 15-60 mg/kg, intragastrically) caused dose-dependent gastric damage after both treatments. Light and electron microscopy revealed that AMG induced minimal changes in the surface epithelium layer, without signs of vasocongestion or leukocytes adherence. AMG (50 mg/kg intragastrically) did not change basal gastric potential difference (PD), whereas acetylsalicylic acid and ibuprofen induced falls in PD of 22 and 27 mV, respectively. AMG (50 mg/kg intragastrically) reduced by 60% the fall in PD induced by 50% ethanol; this inhibition was dependent on the incubation time, and was maximal when AMG was given 4 hr before ethanol. AMG (100 mg/kg intragastrically) induced an increase in NO synthase type 2 (NOS2) activity, which was significantly different from control values, when AMG was administered 4 hr before the test. The metabolites of AMG, tolmetin, MED 5, and guaiacol were ineffective. Pharmacokinetic analysis of the residence time of AMG in the different areas of the gastrointestinal tract, revealed that AMG remains in the gastrointestinal tract at least for 4 hr, the time necessary for a maximal induction of NOS2 and for maximal protection against ethanol-induced damage. In conclusion, these data indicate that the nonsteroidal antiinflammatory drug amtolmetin guacyl is devoid of gastrolesive properties; this gastro-sparing effect seems to involve the production of nitric oxide, which can counteract the damaging effects due to prostaglandin inhibition. The presence in the stomach of the native molecule of amtolmetin guacyl seems to be necessary for the protective effect observed.

PST2238: a new antihypertensive compound that antagonizes the long-term pressor effect of ouabain.

The inhibition of the long-term pressor effect of ouabain may be useful for the therapy of essential hypertension. Here, for the first time, a selective inhibitor of the ouabain pressor effect is described. In vitro, 17beta-(3-furyl)-5beta-androstane-3beta, 14beta, 17alpha-triol (PST 2238) displaced ouabain from its binding sites on purified sodium, potassium ATPase enzyme (Na-K ATPase) (IC50 1.7 x 10(-6) M) without interacting with other receptors involved in blood pressure regulation or hormonal control. In cultured renal cells, incubation with ouabain (10(-10) to 10(-8) M) for 5 days stimulated the Na-K pump at Vmax, whereas PST 2238 showed the same effect at micromolar concentration. The ouabain-dependent increase in the Na-K pump rate was abolished by PST 2238 at concentrations from 10(-14) to 10(-9) M. In rats made hypertensive by chronic infusion of 50 microg/kg/day of ouabain, PST 2238 given p.o at very low doses (0.1-1 microg/kg/day for 4 weeks) abolished the increase in blood pressure and renal Na-K ATPase activity caused by ouabain. PST 2238 did not affect either blood pressure or renal Na-K ATPase activity in normotensive rats. In conclusion, PST 2238 is a very potent compound that normalizes both blood pressure and alterations in the Na-K pump caused by ouabain. Thus it represents the prototype of a new class of antihypertensive drugs that could be effective in forms of hypertension sustained by the concomitant increase of endogenous ouabain levels and alterations in the Na-K pump.