Importance of sphingosine kinase (SphK) as a target in developing cancer therapeutics and recent developments in the synthesis of novel SphK inhibitors.

Sphingosine kinase (SphK) is an oncogenic lipid kinase that regulates the sphingolipid metabolic pathway that has been shown to play a role in numerous hyperproliferative/inflammatory diseases. The SphK isoforms (SphK1 and SphK2) catalyze the conversion of the proapoptotic substrate d-erythrosphingosine to the promitogenic/migratory product sphingosine 1-phosphate (S1P). Accumulation of S1P has been linked to the development/progression of cancer and various other diseases including, but not limited to, asthma, inflammatory bowel disease, rheumatoid arthritis, and diabetic nephropathy. SphK therefore represents a potential new target for developing novel therapeutics for cancer and other diseases. This finding has stimulated the development and evaluation of numerous SphK inhibitors over the past decade or so. In this review, we highlight the recent advancement in the field of SphK inhibitors including SphK1 and SphK2 specific inhibitors. Both sphingolipid based and nolipidic small molecule inhibitors and their importance in treatment of cancer and other diseases are discussed.

Constitutively active mutants of the beta1-adrenergic receptor.

We provide the first evidence that point mutations can constitutively activate the beta(1)-adrenergic receptor (AR). Leucine 322 of the beta(1)-AR in the C-terminal portion of its third intracellular loop was replaced with seven amino acids (I, T, E, F, C, A and K) differing in their physico-chemical properties. The beta(1)-AR mutants expressed in HEK-293 cells displayed various levels of constitutive activity which could be partially inhibited by some beta-blockers. The results of this study might have interesting implications for future studies aiming at elucidating the activation process of the beta(1)-AR as well as the mechanism of action of beta-blockers.

Distribution and subtype determination of beta-receptors in the spinal cord of the adult rat.

1. We determined the number of beta-receptors in the whole spinal cord of the adult rat and in the cervical, thoracal, and lumbal/sacral parts. 2. The undivided spinal cord contains 47 +/- 10 fmol/mg beta-receptors (KD = 2066 +/- 982 pmol/liter), and the cervical part of the spinal cord contains 53 +/- 8 fmol/mg protein (KD = 3224 +/- 1775 pmol/liter). The thoracal part shows 40 +/- 1 fmol/mg protein (KD = 3229 +/- 104 pmol/liter), and the lumbal/sacral spinal cord contains 48 +/- 8 fmol/mg protein (KD = 3610 +/- 1610 pmol/liter). 3. Competitive inhibition studies with l-practolol, dl-atenolol, and ICI 118,551 were performed and we calculated by a computer program in the whole spinal cord the following ratio of beta-receptor subtypes: 80 +/- 5% Beta 1-receptors and 20 +/- 5% beta 2-receptors. 4. The basal and (-)-isoproterenol- and NaF-stimulated activity of adenylate cyclase was highest in the cervical part of the spinal cord and equally distributed between the thoracal and the lumbal/sacral parts. 5. The whole synaptosomal protein of the cervical part of the spinal cord contained 132 +/- 20 fmol, the thoracal part 117 +/- 3 fmol, and the lumbal/sacral part 133 +/- 22 fmol.

Autoradiographic localization of beta-adrenoreceptors in rat uterus.

The inhibitory effects of catecholamines on uterine smooth muscle are known to be mediated through beta-adrenergic receptors. To investigate further the distribution of these receptors in the rat uterus, we utilized in vitro autoradiography using [125I]-cyanopindolol [CYP], a specific beta-receptor ligand that has equal activity for both beta 1- and beta 2-receptor subtypes. The specificity of the labeling and the characterization of receptor subtypes in different cell types were achieved by displacement of radioligand with increasing concentrations of zinterol, a beta-adrenergic agonist with preferential affinity for the beta 2-adrenoreceptor subtype, and practolol, a beta-adrenergic antagonist that binds preferentially to the beta 1-subtype. Quantitative estimation of ligand binding was performed by densitometry. It was shown that the vast majority of beta-adrenoreceptors were of the beta 2-subtype and were found in high concentration not only in the myometrium but also in the endometrial and serosal epithelia. Specific labeling was also observed in glandular elements. These results suggest that beta-adrenoreceptors might be involved in different functions in the uterus.

Studies of practolol on mitochondrial metabolism. Effect on ATPase activity and on membrane potential

Effects of practolol on the ATPase activity and mitochondrial membrane potential were studied. The drug enhanced the ATPase activity on undamagedmitochondria and showed very little effect on broken mitochondria. No effect was observed on the ATPase activity by F1-isolated. Practolol gives rise to a decrease in the membrane potential upon energization of the mitochondrial membrane. It is possible that the mechanism of action of practolol could be related with alterations of in permeability (Na+, K+, or Ca2+)

In vitro pharmacologic activity of congener derivatives and model conjugates of propranolol and practolol.

Previous studies in our laboratory suggested that synthetized derivatives of isoproterenol and histamine could create agonists more potent and receptor and/or tissue selective than the parent compound. In the present study we have evaluated the hypothesis that our results with isoproterenol and histamine derivatives could be extended to include beta-adrenergic antagonists. With this purpose in mind, fourteen derivatives of propranolol and practolol were synthesized and tested in four in vitro systems. The congeners and conjugates were tested using biologic assays (blocking of cAMP accumulation) and/or radioligand binding assays in S-49 lymphoma cells and in rat adipocytes, heart and lung which contain beta 1 and/or beta 2 receptors. Our results indicate that structural modifications distant from the pharmacophore alter the pharmacologic profile of the parent compound. The relative potencies of the derivatives were dependent upon several key factors including the length of the methylene spacer chain and the nature of the substituents on the aromatic ring. The presence of a spacer group with four methylenes resulted in the most active compound in each series when tested on S-49 cells. The derivatives with a paramethyl toluidide group were more potent than the derivatives with a trifluoromethyl toluidide group. The dipeptide derivatives were more potent on adipocyte than S-49 cells, suggesting a preference for beta 1 receptors. Some of the same modifications that led to altered potency and which resulted in an increased receptor and/or tissue selectivity using the progenitors isoproterenol or histamine did extrapolate to the beta blockers. Our data suggest that alterations in receptor and/or tissue selectivity must be imparted by the carrier moiety of the drug and may be related to the biochemical microenvironment of the receptors.

Beta-adrenergic receptors in human trabecular meshwork. Identification and autoradiographic localization.

Beta-adrenergic receptors were identified in slide-mounted sections of human trabecular meshwork by in-vitro labeling, light microscopic autoradiography. Autoradiograms were generated after incubation of slide-mounted tissue sections with 125I-cyanopindolol, a selective high-affinity probe for beta-adrenergic receptors. Experiments in which an excess of either unlabeled Practolol (beta 1 selective ligand) or Zinterol (beta 2 selective ligand) were included suggested that most of the receptors were of the beta 2 subtype. Data from displacement studies using increasing concentrations of the highly specific beta 1- and beta 2-adrenergic receptor antagonists, ICI-89,406 and ICI-118,551, respectively, confirmed the predominance of the beta 2-adrenergic receptors. Similar results obtained with cultured trabecular endothelial cells suggest that the beta-adrenergic receptors may be associated with the endothelial cells of the trabecular meshwork in vivo. These results provide anatomic evidence for the hypothesis that beta-adrenergic agents improve outflow by direct action on the trabecular meshwork and provide a rationale for the development of more selective beta 2-adrenergic agents to increase outflow facility.

Beta-adrenoceptor antagonist activity of bivalent ligands. 1. Diamide analogues of practolol.

Two series of bivalent ligands (P-X-P) containing the (R,S)-3-[(4-aminoaryl)oxy]-1-(isopropylamino)propan-2-ol pharmacophore and a connecting alpha,omega-dicarbonylpoly(methylene) [X = -OC(CH2)nCO-] or alpha,omega-N,N'-bis(carbonylmethylene) polymethylenediamine [X = -OCCH2NH(CH2)nNHCH2CO-] spanner were synthesized and evaluated for beta-adrenoceptor antagonist activity in rat heart and lung membrane preparations. The target compounds were obtained as a mixture of stereoisomers in modest yields by using a three to four step sequence beginning with N-benzylpractolol. The results from the competitive binding studies indicated that binding affinity increased by a factor of up to 160 by increasing the length of the group spanning the pharmacophore moieties. Modest increases in cardioselectivity were also obtained. The data suggest that further increases in spanner length and lipophilicity and optical resolution may improve the potential of a labeled bivalent beta 1-adrenoceptor antagonist to function as a myocardial imaging agent.

Heterogeneity of beta-adrenoreceptors in guinea pig alveolar type II cells.

[3H]Dihydroalprenolol ([3H]DHA) binding to guinea pig alveolar type II cell membrane revealed the presence of both high (KD = 0.38 nM) and low (KD = 4.2 nM) affinity beta-adrenoreceptors. The low affinity site had a higher binding capacity (Bmax = 245.6 fmol/mg protein) than the high affinity site (Bmax = 71.7 fmol/mg protein). Displacement of [3H]DHA by practolol, a selective beta 1 agent, confirmed the existence of two species of adrenoreceptors, corresponding to 21% high affinity (beta 1) and 79% low affinity (beta 2), respectively.

Conclusiveness of rechallenge in the interpretation of adverse drug reactions.

We here consider the extent to which the presumed correlation between an adverse event and the administration of a particular drug can be reinforced by rechallenge. At first question of terminology is: what is a rechallenge? Rechallenge is often accepted too readily as proof of a causal relationship and clinical examples give illustrations of common misinterpretations. Definitions are proposed to characterize: the outcome of rechallenge; the conditions under which rechallenge is performed. In discussing causality, a sharp distinction is drawn between the outcome per se and the establishment of a causal relationship. Finally, the simple concepts proposed here should permit to establish a typology of rechallenge and to assess, by further experimental or retrospective research, the conclusiveness of rechallenge in interpreting adverse drug reactions.

Practolol: aspects of its metabolism and ocular binding in the hamster.

The metabolism and ocular binding of practolol were investigated after oral administration of 14C-practolol to hamsters treated with three modifiers of mixed-function oxidase activity: piperonyl butoxide, cobalt chloride or phenobarbitone. The major urinary metabolites of practolol were 3-hydroxypractolol and polar metabolites which included glucuronide conjugates. A number of unidentified metabolites constituted a minor portion of urinary radioactivity. Each pretreatment modified both the urinary excretion pattern (0-24 h) of practolol and its metabolites and also the metabolite profile of eye extracts 24 h after an oral dose. None of the modifiers of mixed-function oxidase activity had any significant effect on the ocular binding (both extractable and non-extractable components) of practolol and its metabolites. The results indicated that the non-extractable component was neither practolol nor 3-hydroxypractolol.

Synthesis and biodistribution of 125I labeled bivalent analogs of practolol as potential myocardial imaging agents.

Iodinated bivalent ligands 3 and 4 and a monovalent ligand 5 were prepared from the cardioselective beta-antagonist, practolol. 125I-labeled 3, 4 and 5 were prepared by solid phase isotopic exchange reaction with carrier-free Na125I and examined in rats as potential receptor-site-directed myocardial imaging agents. Biodistribution of these agents in rats indicated that 125I-3 and 125I-4 were localized in the heart similarly to 125I-5 and the [125I]iodobenzoyl (6) that was previously reported. Localization of 125I-3 and 125I-4, was more persistent in the heart than that of 125I-monovalent ligands 5 and 6. Heart-to-blood ratios of 125I-3 and 125I-4 were significantly lower than those of 125I-5 and 125I-6, due mainly to slow blood clearance rates of 125I-3 and 125I-4.

The pharmacokinetics of oral and intravenous prenalterol in young, healthy volunteers.

The pharmacokinetics of prenalterol in healthy young volunteers after i.v. and oral dosing has been studied. There is evidence of non-linearity following the i.v. dosing. Evidence of dose-dependent pharmacokinetics following i.v. dosing has been obtained. The sustained-release formulation is very effective: almost 12 h were needed to achieve 50 per cent of the total AUC.

Beta-adrenergic receptor subtypes and subcellular compartmentation of cyclic AMP and cyclic AMP-dependent protein kinase in rabbit cardiomyocytes.

In purified ventricular myocytes from adult rabbit, beta-adrenergic stimulation causes cyclic AMP accumulation and cyclic AMP-protein kinase activation in both particulate and soluble fractions of the cell, whereas prostaglandin E1 elevates cyclic AMP and cyclic AMP-protein kinase activity in the soluble fraction exclusively. Only activation of particulate cyclic AMP-protein kinase activity results in phosphorylase b----a conversion. Using radioligand binding technics, we have determined whether beta 1- and beta 2-receptor subtypes mediate beta-adrenergic effects in particulate and soluble subcellular compartments, respectively. The non-selective antagonist [125I]iodocyanopindolol binds to intact ventricular myocytes with KD of 25 pM and a Bmax of 2.6 X 10(5) receptors/myocyte. Competition for [125I]iodocyanopindolol binding to intact myocytes by the beta-receptor subtype-specific antagonists practolol (beta 1) and zinterol (beta 2) results in monophasic curves with antagonist KD values of 1 microM and 1.5 microM, respectively. We conclude that adult rabbit cardiac myocytes do not possess detectable beta 2 receptors. Further, the ability of isoproterenol to cause elevation of cyclic AMP in two functionally distinct regions within the myocyte must pertain to the actions of a single subtype of beta-receptor, the beta 1-receptor.

Practolol and its metabolites: tissue localization and retention in the hamster.

The disposition of [14C]practolol and its tissue association in the male Syrian hamster were investigated after p.o. administration. After an acute dose of 400 mg/kg, there was a marked persistence in the eye of total radioactivity (primarily practolol; approximate T 1/2 = 7 days) and nonextractable radioactivity which showed minimal decay over 7 days. Nonextractable radioactivity was also found in other tissues (liver, skin, small intestine and skeletal muscle examined). The level of total and nonextractable radioactivity was linearly related to dose (800 mg/kg highest dose examined). There was a marked accumulation of total and nonextractable radioactivity in the eye (7- and 14-fold, respectively) and skin (6- and 7-fold) after daily administration of 400 mg/kg for 22 days. The association of practolol metabolites with tissue macromolecules might explain the immunological changes in patients with the practolol-induced oculomucocutaneous syndrome. The adverse effects of practolol in humans have not yet been reproduced in any laboratory species and so the results must be interpreted with caution in relation to the toxicity of compound.

Pharmacokinetics and plasma-concentration-effect relationships of prenalterol in cardiac failure.

Prenalterol was administered as an intravenous infusion at three incremental rates (60, 120 and 240 nmol/min) to five patients with severe cardiac failure. Haemodynamic, hormonal and metabolic variables were measured at the same time as plasma prenalterol concentrations, and the pharmacokinetics of the drug were studied by following plasma concentrations and urinary excretion during and after the infusion. Concentration-dependent increases in cardiac index, stroke index and stroke work index were observed without increases in arterial pressure, heart rate or myocardial oxygen demand. The reninangiotensin-aldosterone system was stimulated, although the extent of stimulation varied among patients. No strong correlations were found between the logarithm of the plasma prenalterol concentration and effect. Plasma clearance of the drug was lower in cardiac patients than in normal volunteers, but a large decrease in renal clearance was partially balanced by an increase in nonrenal clearance. Over the observed range of concentrations, no deviation from linearity was evident, and plasma concentrations of about 150 nmol/l were effective in improving cardiac function without significant side-effects.

Comparison of the beta 1 selective affinity of prenalterol and corwin demonstrated by radioligand binding.

The ability of the beta 1 partial agonists prenalterol and corwin , to displace the binding of [3H]dihydroalprenolol to the heterogeneous populations of beta-adrenoceptors on rat and rabbit lung membranes was compared. Both drugs exhibited higher affinity for the binding sites in rabbit lung (predominant beta 1) as compared to rat lung (predominant beta 2). Curve fitting analysis of the displacement curves into high affinity (beta 1) and low affinity (beta 2) components indicates that both drugs exhibit beta 1 selective affinity. Further competition experiments performed in the presence of carefully calculated concentrations of highly selective beta 1 (atenolol) or beta 2 (ICI 118,551) antagonists to produce homogeneous receptor subtype populations in rat and rabbit lung membranes respectively, confirmed this beta 1 selective affinity. These results suggest an approximate selective affinity ratio (beta 1 to beta 2) of ten and forty fold for prenalterol and corwin respectively.

Lack of functional influence by prenalterol through alpha 1-adrenoceptors in rat myocardium.

Some of the cardiac properties of the partial beta-agonist prenalterol may indicate a contribution from alpha 1-adrenergic stimulation. We therefore studied whether prenalterol interacted with alpha 1-adrenoceptors in rat myocardium. Combination with propranolol did not reveal an alpha 1-adrenergic inotropic effect of prenalterol in papillary muscles. Neither did prenalterol block the alpha 1-adrenergic response to phenylephrine. In myocardial cells, prenalterol inhibited 3H-prazosin binding to alpha 1-adrenoceptors only at very high concentrations. Prenalterol thus exhibited no functionally important interactions with alpha 1-adrenoceptors in myocardium, and its properties cannot be accounted for in terms of contribution from alpha 1-adrenergic effects.

Selectivity of prenalterol for adrenergic receptor subtypes: a potential mechanism of inotropic selectivity.

To address the hypothesis that myocardial adrenergic receptors mediating chronotropy may be distinguishable from receptors mediating inotropy on the basis of existing subtype classifications, the binding properties of the inotropically selective adrenergic agonist prenalterol to receptor populations of known subtype were characterized. In competitive binding assays, prenalterol exhibited higher affinity for beta 1- than for beta 2-receptors, and a low affinity for both alpha 1- and alpha 2-receptors, in comparison with non-subtype-selective agonists. In addition, log molar displacement curves for prenalterol binding to beta 1-receptors, but not to beta 2- or alpha 2-receptors, were steepened and shifted rightward by guanine nucleotide, suggesting that prenalterol is a beta 1-agonist, but may have antagonist properties at beta 2- and alpha 2-receptors. This receptor subtype profile is similar to that of dobutamine, another inotropically selective adrenergic agonist. This concordance lends further support to the hypothesis that chronotropic responses to exogenous adrenergic agonists may be mediated selectively through beta 2-receptors.