Inhibition of Eph/ephrin interaction with the small molecule UniPR500 improves glucose tolerance in healthy and insulin-resistant mice.

Eph/ephrin interactions and their bidirectional signaling are integral part of the complex communication system between ß-cells, essential for glucose homeostasis. Indeed, Eph/ephrin system was shown to be directly involved in the glucose-stimulated insulin secretion (GSIS) process occurring in the pancreatic islets. Here we tested the Eph antagonist UniPR500 as GSIS enhancer. UniPR500 was validated as EphA5-ephrin-A5 inhibitor in vitro and its efficacy as GSIS enhancer was assessed on EndoC-ßH1 cells. The selectivity of UniPR500 was evaluated by testing this compound on a panel of well-known molecular targets responsible for the regulation of glucose homeostasis. Plasmatic levels of UniPR500 were measured by HPLC/MS approach after oral administration. Finally, UniPR500 was tested as hypoglycemic agent in healthy mice, in a non-genetic mouse model of insulin resistance (IR) and in a non-genetic mouse model of type 1 diabetes (T1D). The compound is an orally bioavailable and selective Eph antagonist, able to increase GSIS from EndoC-ßH1 cells. When tested in vivo UniPR500 showed to improve glucose tolerance in healthy and IR mice. As expected by a GSIS enhancer acting on healthy ß-cells, UniPR500 was ineffective when tested on a non-genetic mouse model of type 1 diabetes, where pancreatic function was severely compromised. In conclusion our findings suggest that Eph targeting is a new and valuable pharmacological strategy in the search of new hypoglycemic agents.

L718Q mutant EGFR escapes covalent inhibition by stabilizing a non-reactive conformation of the lung cancer drug osimertinib.

Osimertinib is a third-generation inhibitor approved for the treatment of non-small cell lung cancer. It overcomes resistance to first-generation inhibitors by incorporating an acrylamide group which alkylates Cys797 of EGFR T790M. The mutation of a residue in the P-loop (L718Q) was shown to cause resistance to osimertinib, but the molecular mechanism of this process is unknown. Here, we investigated the inhibitory process for EGFR T790M (susceptible to osimertinib) and EGFR T790M/L718Q (resistant to osimertinib), by modelling the chemical step (i.e., alkylation of Cys797) using QM/MM simulations and the recognition step by MD simulations coupled with free-energy calculations. The calculations indicate that L718Q has a negligible impact on both the activation energy for Cys797 alkylation and the free-energy of binding for the formation of the non-covalent complex. The results show that Gln718 affects the conformational space of the EGFR-osimertinib complex, stabilizing a conformation of acrylamide which prevents reaction with Cys797.

UniPR129 is a competitive small molecule Eph-ephrin antagonist blocking in vitro angiogenesis at low micromolar concentrations.

BACKGROUND AND PURPOSE: The Eph receptor tyrosine kinases and their ephrin ligands are key players in tumorigenesis and many reports have correlated changes in their expression with a poor clinical prognosis in many solid tumours. Agents targeting the Eph-ephrin system might emerge as new tools useful for the inhibition of different components of cancer progression. Even if different classes of small molecules targeting Eph-ephrin interactions have been reported, their use is hampered by poor chemical stability and low potency. Stable and potent ligands are crucial to achieve robust pharmacological performance. EXPERIMENTAL APPROACH: UniPR129 (the L-homo-Trp conjugate of lithocholic acid) was designed by means of computational methods, synthetized and tested for its ability to inhibit the interaction between the EphA2 receptor and the ephrin-A1 ligand in an elisa binding study. The ability of UniPR129 to disrupt EphA2-ephrin-A1 interaction was functionally evaluated in a prostate adenocarcinoma cell line and its anti-angiogenic effect was tested in vitro using cultures of HUVECs. KEY RESULTS: UniPR129 disrupted EphA2-ephrin-A1 interaction with Ki = 370 nM in an elisa binding assay and with low micromolar potency in cellular functional assays, including inhibition of EphA2 activation, inhibition of PC3 cell rounding and disruption of in vitro angiogenesis, without cytotoxic effects. CONCLUSIONS AND IMPLICATIONS: The discovery of UniPR129 represents not only a major advance in potency compared with the existing Eph-ephrin antagonists but also an improvement in terms of cytotoxicity, making this molecule a useful pharmacological tool and a promising lead compound.

Epidermal growth factor receptor irreversible inhibitors: chemical exploration of the cysteine-trap portion.

Covalent EGFR irreversible inhibitors showed promising potential for the treatment of gefitinib-resistant tumors and for imaging purposes. They contain a cysteine-reactive portion forming a covalent bond with the protein. Irreversible kinase inhibitors have been advanced to clinical studies, mostly characterized by an acrylamide or butynamide warhead. However, the clinical usefulness of these compounds has been hampered by resistances, toxicity and pharmacokinetic problems. Investigation on the structure-activity and structure-reactivity relationships may provide useful information for compounds with improved selectivity and pharmacokinetic properties. This review focuses on the exploration of the cysteine-trap portions able to irreversibly inhibit EGFR and other erbB receptors.

A critical cysteine residue in monoacylglycerol lipase is targeted by a new class of isothiazolinone-based enzyme inhibitors.

BACKGROUND AND PURPOSE: Monoacylglycerol lipase (MGL) is a presynaptic serine hydrolase that inactivates the endocannabinoid neurotransmitter, 2-arachidonoyl-sn-glycerol. Recent studies suggest that cysteine residues proximal to the enzyme active site are important for MGL function. In the present study, we characterize the role of cysteines in MGL function and identify a series of cysteine-reactive agents that inhibit MGL activity with nanomolar potencies by interacting with cysteine residue 208. EXPERIMENTAL APPROACH: A series of cysteine traps were screened for the ability to inhibit MGL in vitro. Rapid dilution assays were performed to determine reversibility of inhibition. Molecular modelling and site-directed mutagenesis were utilized to identify cysteine residues targeted by the inhibitors. KEY RESULTS: The screening revealed that 2-octyl-4-isothiazolin-3-one (octhilinone) inhibited purified rat recombinant MGL (IC(50)= 88 +/- 12 nM) through a partially reversible mechanism. Initial structure-activity relationship studies showed that substitution of the n-octyl group of octhilinone with a more lipophilic oleoyl group increased inhibitor potency (IC(50)= 43 +/- 8 nM), while substitution with a methyl group produced the opposite effect (IC(50)= 239 +/- 68 nM). The inhibitory potency of octhilinone was selectively decreased by mutating cysteine 208 in MGL to glycine (IC(50); wild-type, 151 +/- 17 nM; C208G, 722 +/- 74 nM), but not by mutation of other cysteine residues (C32, C55, C201, C208 and C242). CONCLUSIONS AND IMPLICATIONS: The results indicated that cysteine 208 plays an important role in MGL function and identified a novel class of isothiazolinone-based MGL inhibitors with nanomolar potency in vitro.

Preclinical safety profile of sildenafil.

Sildenafil citrate, marketed as Viagra, for the treatment of erectile dysfunction, has a proven record of safety in humans as predicted by the results of extensive pharmacological and toxicological testing in animals and in vitro, and confirmed by pharmacokinetic exposure data. The aim of this paper is to review succinctly the main findings resulting from these experiments. Daily doses of sildenafil, within and far beyond the human therapeutic range, were given to dogs and rodents for up to 1 and 2 y, respectively. Plasma analyses were conducted to determine the exposure to sildenafil. We found species-specific effects in dogs (Beagle pain syndrome), mice (marked intestinal dilatation) and rats (adaptive reversible hepatocellular hypertrophy associated with secondary thyroid hypertrophy). All these effects in rodents and dogs have no relevance to humans. Morphometric thickness measurements of the retinal layers carried out in response to clinical observations of visual disturbances in humans indicated no difference between treated and control rats and dogs after up to 24 months of treatment. There was no evidence of histopathologic damage to any structures of the visual pathway. Sildenafil had no effects on fertility, no teratogenic potential, was not genotoxic and has no carcinogenic potential. In rats and dogs, safety ratios were 40:1 and 28:1, respectively, in terms of exposure over 24 h (AUC24 h) and 19:1 and 8:1, respectively, in terms of peak plasma concentration (Cmax). These safety ratios illustrate the separation between exposure to sildenafil of animals at large nontoxic doses and the much smaller human therapeutic exposure. This profile highlights the very low risk of human toxicity for sildenafil. The favourable results of the nonclinical safety evaluation of sildenafil in established animal models have been confirmed by many years of clinical experience during the development and marketing of sildenafil.

Echocardiography, a non-invasive method for the investigation of heart morphology and function in laboratory dogs: 1. Method and reference values for M-mode parameters.

We have set up M-mode echocardiographic (EC) recording in beagles in our laboratory and generated reference values for EC indicators of left ventricle function and morphology. Additionally we assessed the effects of sex, strain and body weight on these parameters and the correlation between parameters. M-mode EC under two-dimensional guidance in longitudinal section was performed on 59 male and 49 female beagles from Marshall (USA) and 13 males and 13 females from Harlan (France). The following parameters were measured or calculated: left ventricle internal diameter in diastole and systole (LVIDd and LVIDs), left ventricle and diastolic and end systolic and stroke volumes (EDV, ESV and SV), cardiac output and index (CO and CI), fractional shortening (FS), ejection fraction (EF), the thickness of the septum and left ventricle posterior wall in diastole (STd and LVPWd) and systole (STs and LVPWs), the percentage of thickening of the septum and left posterior wall (PST and PWT), and the mean and maximal velocities of the left ventricle posterior wall (PWVm and PWVM). Heart rate (HR) was measured by cardiac auscultation. Marshall dogs have higher left ventricle dimensions but lower amplitude and velocity of contraction than Harlan dogs. There were also statistically significant differences between sexes for a number of EC parameters mainly those relating to the size of the left ventricle walls or cavity in diastole. Overall these differences were explained by the correlation between these parameters and body weight. Heart rate correlated only with PWVm and PWVM. There were positive correlations between PST, PWT and EF or FS and between velocities and FS or EF. EDV correlates negatively with EF, FS, PST or PWT.

Echocardiography, a non-invasive method for the investigation of heart morphology and function in laboratory dogs: 2. Effects of minoxidil and quinidine on the left ventricle function.

We used echocardiography to investigate the changes in the cardiac function of dogs treated with minoxidil (a vasodilator, administered at doses which can produce mild lesions in the myocardium of the left ventricle) and quinidine (an antiarrhythmic at doses up to 8 times the upper limit of the therapeutic range in dogs). Groups of three beagles received a single administration of minoxidil at doses of 0.5 or 2 mg/kg. Two groups of two dogs received a single administration of quinidine at doses of 80 or 160 mg/kg. Two groups of three control dogs were treated concurrently with the vehicle alone. M-mode echocardiography was performed under two-dimensional echocardiography guidance on three occasions the day before treatment, immediately before dosing and 1, 3 and 24 h after dosing. We measured or calculated end diastolic, end systolic, and stroke volumes (EDV, ESV and SV), fractional shortening (FS), ejection fraction (EF), the percentage of thickening of the septum and of the left ventricle posterior wall (PST and PWT), and the mean and maximal velocities of the left ventricle posterior wall (PWVm and PWVM). At the same time as echocardiography recording, heart rate was measured by cardiac auscultation. Minoxidil produced a marked tachycardia. Less marked increases in heart rate occurred after quinidine. Both compounds were associated with a decrease in ESV and with marked increases in FS, EF, PWVm and PWVM which, in comparison with data for controls, are indicative of an increase in the amplitude and velocity of cardiac contraction. Both drugs also produced a decrease in EDV and consequently there was no increase in SV despite the increased amplitude of ventricular contraction. Cardiac output increased in proportion to the increase in heart rate. Overall, the effects were dose-related and are consistent with the pharmacological properties of the compounds. However, to date these effects have been demonstrated only by invasive methods. To conclude, we have shown that echocardiography allows a non-invasive investigation of the cardiac effects of suprapharmacological doses of antiarrhythmics and of the changes in heart function induced by vasodilators known to cause left ventricular lesions in dogs.

Fat, frail and dying young: survival, body weight and pathology of the Charles River Sprague-Dawley-derived rat prior to and since the introduction of the VAFR variant in 1988.

Trends in survival and body weight were evaluated in 2140 control Sprague-Dawley-derived [Crl: COBS-CD(SD)BR and Crl: COBS-VAF CD(SD)BR] rats used for 24-month rat carcinogenicity studies between 1979 and 1991. Body weight and survival were remarkably stable in the CD-COBS rats used during 1979-1987: at 24 months, the mean survival in males was 68 +/- 5%, and 60 +/- 5% in females. With the CD-COBS-VAF rat, a variant of the CD-COBS strain used between 1988 and 1991, the survival at 24 months dropped to 41 +/- 3% in males, and 44 +/- 7% in females compared to the CD-COBS. The CD-COBS-VAF rat had a significantly reduced life span (P < 0.001 at 24 months), a significant increase in mean body weight (males at 6 months: 672 +/- 24 g vs. 536 +/- 6 g; females: 359 +/- 7 g vs 308 +/- 3 g; P < 0.001) and food consumption (males at 6 months: 31.3 +/- 3.3 vs. 25.4 +/- 2.1 g d-1; females: 22.0 +/- 2.7 g v. 20.3 +/- 2.0 g d-1; P < 0.001). CD-COBS-VAF rats which failed to survive up to study termination had individual body weights at 3, 6 and 12 months which were significantly higher (P < 0.001) than those which survived until 24 months. Our historical data base of control rats (CD-COBS and CD-COBS-VAF) in carcinogenicity studies revealed a significant (males: P < 0.001); females: P < 0.01) and inverse linear relation between mean 3-month body weight and 24-month survival. When compared to CD-COBS animals, CD-COBS-VAF rats showed an increase in the incidence of pituitary tumours in males, mammary fibroadenomas in females, an increase in the incidence of severity of glomerulonephrosis, and a greater incidence of animals which died without any obvious pathology. It is concluded that, in our Sprague-Dawley substrains, both the individual and the group mean body weights in early adult life appear predictive for the individual and group life expectancy. The decrease in longevity in the CD-COBS-VAF rat is principally due to disease and degeneration processes associated with fast growth and high body weight.

The effects of isoproterenol on the isolated perfused rat heart.

In isolated hearts infused with up to 100 ug/ml isoproterenol (ISO), there was only a transient effect on the frequency of contraction, and no effect on buffer flow through the heart. Infusion of hearts with 40-60 or 70-100 ug/ml ISO produced an increase in the release of lactate dehydrogenase, but not creatine kinase. Microscopic examination of hearts revealed myocyte separation and cytoplasmic vacuolation, of similar severity, in both control and ISO infused hearts; there was no necrosis. The data from this experiment are consistent with the view that ISO has little or no direct toxic effect on the heart. Since ISO is cardiotoxic In vivo, it is reasonable to suppose that this toxicity is linked primarily to the pharmacological effects of the drug. However, our study does not exclude the possibility that under the stressed conditions prevailing in vivo following a challenge with ISO, the susceptibility of myocytes to a direct toxic effect of ISO may increase.

Cardiotoxicity of hydralazine and minoxidil in the rat. Influence of age.

In this study, the cardiotoxicity of isoproterenol, hydralazine and minoxidil was compared between young (1.5 months old) and mature (5 months old) rats. Both age classes were also compared for the effect of hydralazine on blood pressure and heart rate. For the 3 compounds, myocardial necrosis was observed and was more marked in mature than in young rats. The age-related increase in sensitivity to cardiotoxic effects which has already been described for isoproterenol, can therefore be extended to hydralazine and minoxidil. Hydralazine produced hypotension and reflex tachycardia. This latter effect appeared to have a longer duration in mature animals, which may be one of the explanation for their increased sensitivity to the cardiotoxic effect of the compound.

Cytochrome P450 in highly purified suspension of nonparenchymal liver cells.

Rat liver nonparenchymal cells (NPC) were prepared by pronase digestion and purified on discontinuous gradients on Nycodenz. Morphological and biochemical characterization of cell suspensions showed that they were free of contamination by hepatocytes. We have confirmed the usefulness of pyruvate kinase activity in monitoring the degree of hepatocyte contamination of NPC and we have derived an equation which allows this carry-over to be calculated. Using highly purified suspensions of NPC we have shown that they contain glucose-6-phosphatase in low but detectable levels. Spectrophotometric studies showed that they contain cytochrome P450, with a specific content of 24 +/- 5 pmole mg-1 cell protein. A potential source of error in previous studies was recognized; namely that peroxidase, present in NPC in high concentration, is able to mask the absorption due to cytochrome P450. Both the presence and inducibility of this enzyme in NPC prepared from rats pretreated with phenobarbital or 3-methylcholanthrene have been confirmed using Western blot analysis.

A comparative study of the effects of ketoconazole and fluconazole on 17-beta estradiol production by rat ovaries in vitro.

In this study we have compared the effects of ketoconazole and fluconazole, a novel triazole antifungal agent, on 17-beta estradiol production in rat ovaries in vitro. For both compounds there was a lag phase, immediately after addition to the test system, during which the rate of oestradiol synthesis remained at control values. This may have been due to the time required for uptake of the compound and transfer to its site of action or for depletion of endogenous pools of intermediates. After the lag phase both compounds produced a reduction in the rate of estradiol synthesis. At any given concentration, fluconazole produced a reduction which was substantially less than that observed with ketoconazole. Indeed 2 microM ketoconazole reduced the rate of oestradiol production by greater than 90% while 10 microM fluconazole caused only a 70% reduction. These findings are consistent with reports that these compounds are inhibitors of cytochrome P450 and with the reduced sensitivity of mammalian cytochrome P450 to fluconazole as compared with ketoconazole.

A comparative study of 1-substituted imidazole and 1,2,4-triazole antifungal compounds as inhibitors of testosterone hydroxylations catalysed by mouse hepatic microsomal cytochromes P-450.

Three imidazole antifungal agents, ketoconazole, miconazole and tioconazole, and a group of structurally related 1-substituted imidazole and 1,2,4-triazole compounds were evaluated as inhibitors of the oxidative metabolism of testosterone catalysed by mouse hepatic microsomal cytochromes P-450. Spectroscopic studies showed that both imidazoles and triazoles interacted with ferric cytochrome P-450 in hepatic microsomes to produce type II difference spectra which could be distinguished by their different absorbance maxima; 429-430 nm and 425-426 nm respectively. Compound 4, which possesses both types of functional group, produced a spectrum which resembled that of imidazole compounds, indicating that the imidazole moiety had a higher affinity than the triazole for the haem of cytochromes P-450 present in microsomes. The test compounds differentially inhibited regio- and stereo-specific testosterone metabolism and the pattern of inhibition varied with the 1-substituent on the azole ring. Ketoconazole was a potent inhibitor of testosterone 6 beta-hydroxylation (IC50 0.08 microM) but was considerably less active against other hydroxylations and 17 beta-oxidation to androstenedione (IC50 range 13 to greater than 100 microM). In contrast, tioconazole (IC50 range 0.18 to 3.3 microM) and miconazole (IC50 range 0.15 to 10 microM) were relatively non-selective. Compounds 1 and 2, which differed from each other only in the type of azole ring, were most active against 16 beta-hydroxylation. The triazole analogue (compound 2) was a significantly more potent inhibitor of 16 beta-hydroxylation than the imidazole (compound 1), equipotent against androstenedione formation and less active against the other hydroxylations. Two relatively polar bis-azole analogues (compounds 3 and 4) were most active against androstenedione formation; however, in general they were less inhibitory than the lipophilic azoles. We conclude that azole antifungal agents of differing structure show different patterns of selective interaction with cytochromes P-450, a phenomenon primarily dependent on the 1-substituent on the azole ring, but also modulated to a lesser extent by the type of azole ring (imidazole or triazole).

Testosterone metabolite profiles reveal differences in the spectrum of cytochrome P-450 isozymes induced by phenobarbitone, 2-acetylaminofluorene and 3-methylcholanthrene in the chick embryo liver.

The regiospecificity and stereoselectivity of testosterone hydroxylation by hepatic microsomes prepared from control, PB, 3MC and 2AAF treated chick embryos has been analysed. Microsomes prepared from control animals hydroxylate testosterone at the 16 alpha and 6 beta positions exclusively: 3MC treatment only causes comparatively minor alterations in the rates of these conversions. PB and 2AAF treatment induced 16 beta-hydroxylation, whilst only 2AAF caused a substantial induction of 6 beta hydroxylation. This data suggests that in the chick, 2AAF not only induces P-450 subforms which are also induced by PB but additional subforms which are not markedly induced by either PB or 3MC.

Developmental changes in the rate of production of an unusual testosterone metabolite, 4-androstene-3 alpha, 17 beta-diol, by chick liver microsomes.

Testosterone is produced by the chick embryo testis from the 13th day of incubation. We have investigated the ability of microsomes prepared from the fetal and neonatal liver to metabolize testosterone and have found that the principal metabolite generated by microsomes in the presence of NADPH is 4-androstene-3 alpha, 17 beta-diol. The rate of production of this metabolite declined sharply over the time of hatching. Conversely, 16 alpha-hydroxytestosterone production increased transiently just after hatching. Our findings indicate that chick liver microsomes contain a 3 alpha-hydroxysteroid dehydrogenase (3 alpha-hydroxysteroid: NAD(P) oxidoreductase, EC 1.1.1.50) whose activity changes during development.

A study into the effects of 2-acetylaminofluorene on hepatic monooxygenase activities in the chick embryo.

2AAF is a potent inducer of cytochrome P-450 in the chick embryo liver. The induction has been characterized with respect to a range of monooxygenase activities and the regiospecificity of 2AAF hydroxylation. Similarities to the response elicited by both PB and 3MC were noted. 2AAF was rapidly deacetylated by hepatic microsomes prepared from control animals to 2AF, an inhibitor of monooxygenase activity. Metabolites generated in vivo and carried over in vitro might have therefore interfered with the subsequent kinetic analysis. In general terms induction of a unique cytochrome P-450 subform(s) could not be attributed to 2AAF in the chick embryo. The data is discussed with respect to the reported resistance of avian species to the hepatocarcinogenic effects of 2AAF. Two possibilities are highlighted, a diversion of 2AAF to ring hydroxylated metabolites and/or deacetylation of 2AAF. Both effects could reduce carcinogenicity by decreasing the concentration of proximate carcinogen and/or promoter(s).

A characterization of haem uptake and intracellular distribution by isolated hepatocytes.

The uptake and intracellular distribution of haem by isolated rat hepatocyte suspensions was studied. An increase in cell haem content occurred after a challenge with 5, 10 or 20 microM haem, supplied as methaemalbumin. The rate of haem uptake was temperature dependent; no non-specific binding occurred. Intracellular haem distribution data are consistent with a rapid association of haem with the endoplasmic reticulum fraction prior to its accumulation in the cytosol and at the mitochondrion.