Prevalence of herpes simplex virus type 1 glycoprotein G (gG) and gI genotypes in patients with herpetic keratitis.

AIM: Recent phylogenetic analyses on the herpes simplex virus type 1 (HSV-1) genes US4, encoding glycoprotein G (gG) and US7, encoding gI, of clinical HSV-1 isolates have led to the classification of HSV-1 into three genotypes, arbitrarily designated as A, B and C. The prevalence of the HSV-1 gG and gI genotypes and their potential disease association was determined in a large cohort of patients with herpetic keratitis (HK). METHODS: Primary corneal HSV-1 isolates of 178 HK patients were genotyped by a PCR-based restriction fragment length polymorphism method targeting the viral genes US4 and US7. RESULTS: Genotype B was more frequently expressed by the corneal HSV-1 isolates compared with genotypes A and C. Fifty-five of 178 corneal isolates (31%) had different genotypes in both loci. No clinically relevant associations were observed between the HSV-1 genotypes and disease outcome in the HK patients studied. CONCLUSIONS: The data presented demonstrate a high frequency of recombinant corneal HSV-1 isolates and suggest that clinical outcome of HSV-1-induced keratitis is independent of a gG or gI genotype.

R306465 is a novel potent inhibitor of class I histone deacetylases with broad-spectrum antitumoral activity against solid and haematological malignancies.

R306465 is a novel hydroxamate-based histone deacetylase (HDAC) inhibitor with broad-spectrum antitumour activity against solid and haematological malignancies in preclinical models. R306465 was found to be a potent inhibitor of HDAC1 and -8 (class I) in vitro. It rapidly induced histone 3 (H3) acetylation and strongly upregulated expression of p21waf1,cip1, a downstream component of HDAC1 signalling, in A2780 ovarian carcinoma cells. R306465 showed class I HDAC isotype selectivity as evidenced by poor inhibition of HDAC6 (class IIb) confirmed by the absence of downregulation of Hsp90 chaperone c-raf protein expression and tubulin acetylation. This distinguished it from other HDAC inhibitors currently in clinical development that were either more potent towards HDAC6 (e.g. vorinostat) or had a broader HDAC inhibition spectrum (e.g. panobinostat). R306465 potently inhibited cell proliferation of all main solid tumour indications, including ovarian, lung, colon, breast and prostate cancer cell lines, with IC50 values ranging from 30 to 300 nM. Haematological cell lines, including acute lymphoblastic leukaemia, acute myeloid leukaemia, chronic lymphoblastic leukaemia, chronic myeloid leukaemia, lymphoma and myeloma, were potently inhibited at a similar concentration range. R306465 induced apoptosis and inhibited angiogenesis in cell-based assays and had potent oral in vivo antitumoral activity in xenograft models. Once-daily oral administration of R306465 at well-tolerated doses inhibited the growth of A2780 ovarian, H460 lung and HCT116 colon carcinomas in immunodeficient mice. The high activity of R306465 in cell-based assays and in vivo after oral administration makes R306465 a promising novel antitumoral agent with potential applicability in a broad spectrum of human malignancies.

Effect of an hdm-2 antagonist peptide inhibitor on cell cycle progression in p53-deficient H1299 human lung carcinoma cells.

The hdm-2 oncogene is overexpressed in several types of malignancies including osteosarcomas, soft tissue sarcomas and gliomas and hdm-2 has been associated with accelerated tumor formation in both hereditary and sporadic cancers. Among the other key binding partners, hdm-2 forms a complex with the tumor suppressor p53, resulting in a rapid proteasome-mediated degradation of the p53 protein. This positions the hdm-2-p53 complex as an attractive target for the development of anticancer therapy and recently the first small molecule hdm-2 antagonist has been reported. Development of hdm-2 antagonists is currently focused on malignancies containing a wild-type p53 genotype, which is the case in approximately half of human cancer indications. However, hdm-2 has also been implicated in oncogenesis in the absence of p53. We therefore studied the effect of hdm-2 antagonists in p53-deficient human H1299 lung carcinoma cells. The hdm-2 antagonistic peptide caused G1 cell cycle arrest, inhibited colony growth and induced expression of G1 checkpoint regulatory proteins, such as p21(waf1,cip1). These data demonstrate that hdm-2 regulates the G1 cell cycle checkpoint in a p53-independent manner, suggesting that hdm-2 antagonists represent a novel class of anticancer therapeutics with broad applicability towards tumors with different p53 genetic backgrounds.

Inhibition of all-TRANS-retinoic acid metabolism by R116010 induces antitumour activity.

All-trans-retinoic acid is a potent inhibitor of cell proliferation and inducer of differentiation. However, the clinical use of all-trans-retinoic acid in the treatment of cancer is significantly hampered by its toxicity and the prompt emergence of resistance, believed to be caused by increased all-trans-retinoic acid metabolism. Inhibitors of all-trans-retinoic acid metabolism may therefore prove valuable in the treatment of cancer. In this study, we characterize R116010 as a new anticancer drug that is a potent inhibitor of all-trans-retinoic acid metabolism. In vitro, R116010 potently inhibits all-trans-retinoic acid metabolism in intact T47D cells with an IC(50)-value of 8.7 nM. In addition, R116010 is a selective inhibitor as indicated by its inhibition profile for several other cytochrome P450-mediated reactions. In T47D cell proliferation assays, R116010 by itself has no effect on cell proliferation. However, in combination with all-trans-retinoic acid, R116010 enhances the all-trans-retinoic acid-mediated antiproliferative activity in a concentration-dependent manner. In vivo, the growth of murine oestrogen-independent TA3-Ha mammary tumours is significantly inhibited by R116010 at doses as low as 0.16 mg kg(-1). In conclusion, R116010 is a highly potent and selective inhibitor of all-trans-retinoic acid metabolism, which is able to enhance the biological activity of all-trans-retinoic acid, thereby exhibiting antitumour activity. R116010 represents a novel and promising anticancer drug with an unique mechanism of action.

Induction of the oxidative catabolism of retinoid acid in MCF-7 cells.

Cytochrome P450-dependent oxidation is a pathway for all-trans-retinoic acid (all-trans-RA) catabolism. Induction of this catabolic pathway was studied in MCF-7 breast cancer cells. MCF-7 cells showed low constitutive all-trans-RA catabolism. Concentration-dependent induction was obtained by preincubation of the cells with all-trans-RA (10(-9) to 10(-6) M). Onset of induction was fast, being detectable within 60 min, with maximal induction (45-fold) obtained after 16 h. Enzymatic characterization of induced all-trans-RA catabolism showed an estimated Km value (Michaelis-Menten constant) of 0.33 microM and a Vmax value (maximal velocity of an enzyme-catalysed reaction) of 54.5 fmol polar all-trans-RA metabolites 10(6) cells(-1) h(-1). These kinetic parameters represent the overall formation of polar metabolites from all-trans-RA. Induction of all-trans-RA catabolism was also obtained with other retinoids, CH55 >> 13-cis-RA = all-trans-RA > 9-cis-RA > 4-keto-all-trans-RA > 4-keto-13-cis-RA > retinol. The potency of the retinoids to induce all-trans-RA catabolism was correlated to their retinoic acid receptor affinity (Crettaz et al, 1990; Repa et al, 1990; Sani et al, 1990). Induction of all-trans-RA catabolism was inhibited by actinomycin D. Furthermore, all-trans-RA did not increase cytosolic retinoic acid-binding protein (CRABP) mRNA levels. These data suggest that induction of all-trans-RA catabolism in MCF-7 cells is a retinoic acid receptor-mediated gene transcriptional event. Induced all-trans-RA catabolism was inhibited by various retinoids with decreasing potency in the order: all-trans-RA > 4-keto-all-trans-RA > 13-cis-RA > 9-cis-RA > 4-keto-13-cis-RA > retinol > CH55. The antitumoral compound liarozole-fumarate inhibited all-trans-RA catabolism with a potency similar to that of all-trans-RA.

Effects of liarozole, a new antitumoral compound, on retinoic acid-induced inhibition of cell growth and on retinoic acid metabolism in MCF-7 human breast cancer cells.

Liarozole is a new imidazole derivative with antitumoral properties. Effects of the compound alone and in combination with all-trans-retinoic acid on proliferation of MCF-7 human breast cancer cells were examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Following 9 days of drug exposure, MCF-7 cell growth was concentration dependently inhibited by all-trans-retinoic acid (drug concentration resulting in 50% growth inhibition, 2 x 10(-8) M), while liarozole at 10(-5) M inhibited cell growth by only 35%. When MCF-7 cells were incubated with a combination of all-trans-retinoic acid and liarozole, the antiproliferative effect of all-trans-retinoic acid was clearly enhanced. This enhancement was dependent on the liarozole concentration and was more than 10-fold. A combination of 10(-8) M all-trans-retinoic acid and 10(-6) M liarozole resulted in a greater antiproliferative effect than that obtained with 10(-7) M all-trans-retinoic acid alone. When MCF-7 cells were incubated for 4 h with [3H]all-trans-retinoic acid, the radioactivity in the supernatant consisted of unaltered retinoid. However, when cells had been pretreated with 10(-6) M all-trans-retinoic acid overnight, they were able to substantially metabolize [3H]all-trans-retinoic acid during a subsequent 4-h incubation. High-performance liquid chromatography analysis of the supernatants revealed that the reaction products consisted mainly of very polar metabolites. Liarozole inhibited the metabolism of all-trans-retinoic acid in MCF-7 cells with 10(-5) M liarozole reducing the amount of polar metabolites by 87%. It is concluded that the enhancement by liarozole of the antiproliferative effects of retinoic acid on MCF-7 human breast cancer cells is probably due to inhibition of retinoic acid metabolism. Further research into these effects in MCF-7 cells as well as in other cancer cell lines will provide more information concerning the exact mechanism of action of liarozole and the use of inhibitors of retinoid metabolism in cancer treatment.

Comparative effects of the aromatase inhibitor R76713 and of its enantiomers R83839 and R83842 on steroid biosynthesis in vitro and in vivo.

R76713 (6-[(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-methyl-1H- benzotriazole) is a selective, non-steroidal aromatase inhibitor containing an asymmetric carbon atom. In this paper, we compare the effects of R76713 (racemate) with its enantiomers R83839 (the levo-isomer) and R83842 (the dextro-isomer) on steroid biosynthesis in rat cells in vitro and in the rat in vivo. In rat granulosa cells, aromatase activity was inhibited by 50% at concentrations of 0.93 nM of R76713, 240 nM of R83839 and 0.44 nM of R83842, revealing a 545-fold difference in activity between both enantiomers. Up to 1 microM, none of the compounds had any effect on steroid production in primary cultures of rat testicular cells. Above this concentration all three compounds showed a similar slight inhibition of androgen synthesis with a concomitant increase in the precursor progestins, indicative for some effect on the 17-hydroxylase/17,20-lyase enzyme. In rat adrenal cells none of the compounds showed any effect on corticosterone synthesis. At concentrations above 1 microM there was an increase in the levels of 11-deoxycorticosterone pointing towards an inhibition of the 11-hydroxylase enzyme. This increase was more pronounced for R83839 than for R76713 and R83842. In vivo, in PMSG-primed rats, R83842 reduced plasma estradiol by 50%. 2 h after oral administration of 0.0034 mg/kg, whereas 0.011 mg/kg of R76713 and 0.25 mg/kg of R83839 were needed to obtain the same result. Oral administration of up to 20 mg/kg of the compounds did not significantly affect plasma levels of adrenal steroids in LHRH/ACTH-injected rats. Plasma testosterone was lowered at 10 and 20 mg/kg of R83842 and at the highest dose (20 mg/kg) of R76713 and R83839. In conclusion, the present study shows that the aromatase inhibitory activity of R76713 resides almost exclusively in its dextro-isomer R83842. R83842 exhibits a specificity for aromatase as compared to other enzymes involved in steroid biosynthesis of at least a 1000-fold in vitro as well as in vivo. This confirms the extreme selectivity previously found for the racemate.

R 76713, a new specific non-steroidal aromatase inhibitor.

The effects of R 76713, a new triazole derivative, on rat ovarian, testicular and adrenal steroidogenesis were investigated both in vitro and in vivo. In vitro R 76713 is a very potent inhibitor of the aromatase enzyme in rat granulosa cells, showing an IC50-value of 3.0 +/- 0.2 nM. The compound is about 1000 times more active than aminoglutethimide which shows an IC50-value of 3900 +/- 2800 nM in the same system. R 76713 is also a highly selective aromatase inhibitor. In cultures of ovarian, testicular and adrenal cells, formation of progesterone, androgens and glucocorticoids was only affected by drug concentrations higher than 1 microM. In vivo, single oral drug doses of 0.05 mg/kg lowered plasma estradiol levels of PMSG-primed female rats by more than 90%. An ED50-value of 0.005 mg/kg could be calculated. A single oral dose of 1 mg/kg suppressed plasma estradiol levels almost completely for 24 h. A dose of 0.1 mg/kg lowered plasma estradiol by more than 90% for 8 h. In vivo, R 76713 also showed a highly selective profile. In LHRH/ACTH-injected rats, plasma levels of testicular and adrenal steroids remained unchanged after administration of a drug dose of 20 mg/kg. R 76713 at drug concentrations of 10 microM, showed no interaction in vitro with estrogen-, progestin-, androgen- and glucocorticoid-receptors. Given orally at 20 mg/kg for 3 days the compound also showed no estrogen or androgen agonistic or antagonistic effects.

Aromatase inhibition by the antifungal ketoconazole.

The aromatase inhibitory properties of the antifungal ketoconazole were compared with those of aminoglutethimide. In rat granulosa cells ketoconazole and aminoglutethimide showed IC50 values for aromatase inhibition of 2 X 10(-6) and 6 X 10(-7) M respectively. In the rat, in vivo, ketoconazole was 5 times less potent than aminoglutethimide. In young women, 400 mg of ketoconazole only marginally lowered plasma levels of estradiol-17 beta. It is concluded that ketoconazole is not a compound of choice for clinical use as an aromatase inhibitor.

Identification of the serotonin-S2 receptor ligand binding site by photoaffinity labelling with 7-azido-8-[125I]ketanserin ([125I]AZIK).

7-Azido-8-[125I]ketanserin ([125I]AZIK) was characterized as a potent photoaffinity probe for serotonin-S2 receptors. In reversible binding experiments, [125I]AZIK bound with high affinity (Kd = 0.69 nM) to rat frontal cortex membranes. When incubation with [125I]AZIK was followed by UV irradiation, the binding was found to be irreversible. Protection experiments with various drugs demonstrated the serotonin-S2 nature of the photoaffinity labelling. SDS-polyacrylamide gel electrophoresis of the photolabelled membranes allowed one to identify the serotonin-S2 receptor ligand binding site as a single polypeptide with a molecular mass of approx. 67,500 Da. [125I]AZIK will be a valuable tool for the elucidation of the serotonin-S2 receptor structure.

In vitro labeling of serotonin-S2 receptors: synthesis and binding characteristics of [3H]-7-aminoketanserin.

[3H]-7-Aminoketanserin (7-amino-3-[2-[4-(2-tritio-4-fluorobenzoyl)-1- piperidinyl]ethyl]-2,4-(1H,3H)-quinazolinedione), an amino derivative of the selective serotonin-S2 antagonist ketanserin, was synthesized and tested for in vitro labeling of serotonin-S2 receptors. The compound showed a very high affinity for both membrane-bound and detergent-solubilized serotonin-S2 receptors with KD values of 0.35 and 2.03 nM, respectively. At nanomolar concentrations, binding to serotonin-S1 sites was totally absent. Serotonin-S2 receptor binding was characterized by a slow dissociation and a very low nonspecific binding. In rat frontal cortex preparations, binding could be displaced by nanomolar concentrations of different serotonin antagonists and micromolar concentrations of serotonin agonists. Compounds with other pharmacological profiles were poorly or not active. Introduction of an amino function in this new radioligand led to a decreased lipophilicity. Therefore, besides being a valuable radioligand for routine binding studies, [3H]-7-aminoketanserin will probably be a good ligand for labeling serotonin-S2 receptors on intact cells.

Solubilization of rat brain serotonin-S2 receptors using CHAPS/salt.

Serotonin-S2 receptors from rat frontal cortex were solubilized with a mixture of 6.8 mM CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate) and 1.4 M sodium chloride. This new solubilization procedure solubilized about 40% of the membrane-bound serotonin-S2 receptors in an active form. The solubilized receptors were not sedimented after 1 h of centrifugation at 100 000 X g and they passed freely through 0.20 micron filters. The solubilized preparation showed high affinity binding of [3H]ketanserin and revealed a typical serotonin-S2 receptor profile: binding could be displaced by nanomolar concentrations of different serotonin antagonists and by micromolar concentrations of serotonin agonists. Compounds belonging to other pharmacological classes were poorly, or not active. Upon density gradient sedimentation, Svedberg coefficients of approximately 5 S were found on sucrose gradients made with H2O or D2O as the solvent. This was much lower than the value of 11.5 S previously reported from lysophosphatidylcholine-solubilized serotonin-S2 receptors.

Photoaffinity probes for serotonin and histamine receptors. Synthesis and characterization of two azide analogues of ketanserin.

Two azide analogues of ketanserin (6- and 7-azido-3-[2- [4-(4-fluorobenzoyl)-1-piperidinyl]ethyl]-2, 4(1H,3H)-quinazolinedione) were synthesized and tested as possible photoaffinity probes for serotonin-S2 and histamine-H1 receptors. In reversible binding experiments, the azides showed high affinity for both receptor types. When membrane preparations were incubated with nanomolar concentrations of 7-azidoketanserin and subsequently irradiated with UV light, both serotonin and histamine receptors became irreversibly blocked. This irreversible binding was dependent on azide concentrations and time of irradiation and did not change in the presence of the scavenger p-aminobenzoic acid. In contrast, irreversible blockade at low concentrations of 6-azidoketanserin was only obtained for histamine receptors. However, this blockade was abolished by addition of the scavenger p-aminobenzoic acid indicating that it was not due to a real photoaffinity mechanism. In the rat prefrontal cortex, irreversible blocking of serotonin receptors with 7-azidoketanserin could be inhibited by serotonin agonists or antagonists but not by histaminergic compounds. On the contrary, in the guinea pig cerebellum, inactivation of histamine receptors could be inhibited by histamine antagonists and histamine itself but not by serotonergic compounds. This provides a way for differential photolabeling of either of these receptors.

Differential photoaffinity labelling of serotonin-S2 receptors and histamine-H1 receptors using 7-azidoketanserin.

7-Azidoketanserin, a potent photoaffinity probe for serotonin-S2 receptors was shown to irreversibly photoinactivate histamine-H1 receptors as well. The photolabelling of H1-receptors could be prevented by several selective histamine-H1 antagonists. In guinea pig cerebellum, a brain area that is highly enriched in H1-receptors, photolabelling could be directed exclusively to these receptors by adding a high concentration of pipamperone, which selectively blocks serotonin-S2 receptors. In rat pre-frontal cortex, a region that is enriched in serotonin-S2 receptors, pyrilamine was used to block H1-receptors, thereby directing the photolabelling exclusively to S2-receptors.

Photoaffinity labelling of dopamine receptors. Synthesis and binding characteristics of azapride.

A new compound, azapride, i.e. 4-azido-5-chloro-2-methoxy-N-[1-(phenylmethyl)-4-piperidinyl]benza mide, the azide derivative of the dopamine antagonist clebopride, was synthesized and tested for its usefulness as a photoaffinity probe for dopamine receptors. Without photolysis the azide behaved as a very selective dopamine antagonist. When a microsomal preparation of dog striatum was pretreated with the azide and subsequently irradiated with ultraviolet light, dopamine receptors became irreversibly blocked. This reaction was dependent on both azide concentration and time of irradiation. The irradiation-induced inhibition was quite selective for the dopamine receptor; various other receptor systems were not affected. Moreover the irreversible inhibition of dopamine receptors from dog striatum could be prevented by dopamine antagonists as well as by a dopamine agonist, but not by a serotonin antagonist. The irradiation-induced inactivation by azapride remained unchanged in the presence of the scavenger p-aminobenzoic acid. These findings provide evidence that azapride labels dopamine receptors specifically and irreversibly and by a true photoaffinity mechanism. It should therefore be of great value for further molecular characterization and purification of dopamine receptors.

Characterization of cholate-solubilized dopamine receptors from human, dog and rat brain.

[3H]Spiperone binding sites were solubilized in high yield from human, dog and rat brain with a mixture of sodium cholate (0.3% w/v) and sodium chloride (1.4 M). The binding sites were not sedimented after one hour at 100,000 g, they passed freely through 0.20 micron filters, migrated as a single peak in gradient sedimentation and were retarded upon gel filtration, proving that they were truly solubilized. The solubilized binding sites were definitely of dopaminergic nature. They showed saturable, reversible, high affinity binding of [3H]spiperone; displacement of [3H]spiperone binding by nanomolar concentrations of dopamine antagonists and micromolar concentrations of serotonin antagonists; stereo-specificity and a good correlation with drug affinities for membrane preparations. The non-displaceable, non-specific [3H]spiperone binding was very low. Gradient sedimentation analysis revealed a sedimentation coefficient of 12 S for dog solubilized preparations, 9 S for rat solubilized preparations and only 2.5 S for human solubilized preparations (values, uncorrected for detergent binding). Gel filtration experiments seem to confirm these molecular characteristics. Therefore the present results show that the dopamine receptor reveals the same pharmacological properties when solubilized with cholate-salt from rat, dog or human brain, while physico-chemical properties seem to indicate some differences.