Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression.

Mutated ras genes are found in a large number of human tumors and, therefore, constitute one of the primary targets for cancer treatment. Microinjection of the neutralizing anti-Ras monoclonal antibody Y13-259 was previously reported to induce transient phenotypic reversion of ras-transformed rodent fibroblasts in vitro. We have prepared a single-chain Fv fragment (scFv) derived from Y13-259, and here, we show that intracellular expression of the scFv led to the specific inhibition of the Ras signaling pathway in Xenopus laevis oocytes and NIH3T3 fibroblasts. Moreover, neutralizing Ras with the scFv specifically promoted apoptosis in vitro in human cancer cells but not in untransformed cells. As a step toward cancer gene therapy, we finally demonstrated that intratumor transduction of HCT116 colon carcinoma cells with the anti-Ras scFv using an adenoviral vector elicited sustained tumor regression in nude mice.

The pan phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) blocks survival, adhesion and proliferation of primary chronic lymphocytic leukemia cells.

The phosphoinositide 3-kinases (PI3Ks) are critical components of the B-cell receptor (BCR) pathway and have an important role in the pathobiology of chronic lymphocytic leukemia (CLL). Inhibitors of PI3Kδ block BCR-mediated cross-talk between CLL cells and the lymph node microenvironment and provide significant clinical benefit to CLL patients. However, the PI3Kδ inhibitors applied thus far have limited direct impact on leukemia cell survival and thus are unlikely to eradicate the disease. The use of inhibitors of multiple isoforms of PI3K might lead to deeper remissions. Here we demonstrate that the pan-PI3K/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) was more pro-apoptotic to CLL cells--irrespective of their ATM/p53 status--than PI3Kα or PI3Kδ isoform selective inhibitors. Furthermore, SAR245409 blocked CLL survival, adhesion and proliferation. Moreover, SAR245409 was a more potent inhibitor of T-cell-mediated production of cytokines, which support CLL survival. Taken together, our in vitro data provide a rationale for the evaluation of a pan-PI3K inhibitor in CLL patients.

Cancer gene therapy mediated by CTS1, a p53 derivative: advantage over wild-type p53 in growth inhibition of human tumors overexpressing MDM2.

Recently, a new p53 derivative has been designed, namely chimeric tumor suppressor 1 (CTS1), in which the p53 domains that are known to mediate p53 inactivation have been replaced. In this study, the antitumoral activity of CTS1 mediated by adenovirus vector has been evaluated in comparison with a p53 adenovirus vector in various human tumor cell lines. In vitro, in terms of cell growth inhibition, the CTS1 vector was significantly (P < .01) more efficient (2- to 7-fold) than the p53 vector in tumor models overexpressing an inhibitor of p53, murine double minute-2. This result was confirmed in vivo in a pre-established tumor developed in nude mice. In an osteosarcoma model overexpressing murine double minute-2, we have shown a significantly (P < .05) higher tumor growth delay with the CTS1 vector compared with the p53 vector (25.6 days compared with 12.4 days). Furthermore, both in vitro and in vivo, we have shown that this higher inhibition of tumor growth with the CTS1 vector was correlated with a higher induction of apoptosis. Therefore, CTS1 is a potentially improved tumor suppressor gene for the treatment of human tumors resistant to wild-type p53 gene therapy.

N6-substituted adenosine receptor agonists. Synthesis and pharmacological activity as potent antinociceptive agents.

Novel N6-(indol-3-yl)alkyl derivatives of adenosine were synthesized. The adenosine receptor affinity and the antinociceptive activity of these compounds were assessed in binding studies and the phenylbenzoquinone-induced writhing test. Most of these analogues exhibited a potent analgesic activity without side effects. Among them, compound 3c (UP 202-32) bound to A1 (Ki = 110 nM) and A2 (Ki = 350 nM) adenosine receptors in a specific manner since it did not interact with many other receptors, especially opioid binding sites. The antinociceptive activity in the phenylbenzoquinone assay (ED50 = 3.3 mg/kg po) was antagonized by 8-cyclopentyltheophylline, suggesting that an adenosinergic mechanism underlies the analgesic activity observed with this compound. The data obtained with these new N6-substituted adenosine receptor agonists emphasize the interest of such compounds in the treatment of pain.

Synthesis and SAR studies of novel triazolopyrimidine derivatives as potent, orally active angiotensin II receptor antagonists.

The synthesis and pharmacological activity of new nonpeptide angiotensin II (AII) receptor antagonists are presented. These [1,2,4]-triazolo[1,5-c]pyrimidine and 1,2,4-triazolo[4,3-c]pyrimidine derivatives represent a new class of bicyclic antagonists that produced a potent, oral antihypertensive activity in the renal artery-ligated rat model. In vitro, they displayed a high affinity for rat adrenal AII receptors and were found to be specific for the AT1 receptor subtype. A SAR study has shown the importance of the 8-[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl for oral activity and the critical role of alkyl substituents at 5- and 7-positions. No significant differences were found between the [1,5-c] and [4,3-c]series. UP 269-6 (5-methyl-7-n-propyl-8-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl ]- [1,2,4]-triazolo[1,5-c]pyrimidin-2(3H)-one, derivative 29) was selected as the lead compound. It was shown to be a highly potent antihypertensive derivative (decrease in mean arterial pressure of 39.6 +/- 7.2 mmHg at 1 mg/kg po in renal artery-ligated rat) with a long duration of action which displayed a high affinity for adrenal AII receptors with a marked selectivity for the AT1 receptor subtype (Ki AT1 = 24 nM; Ki AT2 = 79,200 nM). This compound is currently undergoing extensive pharmacological and clinical development.

In vitro and in vivo effects of UP 269-6, a new potent orally active nonpeptide angiotensin II receptor antagonist, on vascular smooth muscle cell proliferation.

1. The present studies were designed to measure the affinity of UP 269-6, a newly developed angiotensin AT1 receptor antagonist, for vascular AT1 receptors from normotensive and hypertensive rats and to investigate in vitro, its effects on angiotensin II (AII)-induced hyperplasia and hypertrophy of vascular smooth muscle cells (VSMC). In addition the in vivo effects of UP 269-6 on neointimal proliferation in a carotid artery balloon injury in normotensive rats were also investigated. 2. UP 269-6 selectively inhibited [125I]-Sar1-Ile8-AII binding to vascular AT1 receptors present on VSMC derived from normotensive Wistar rat and from SHR (Ki = 16.6 +/- 3.6 nM and 7.5 +/- 2.0 nM, respectively). In comparison, losartan and its metabolite, EXP 3174, inhibited [125I]-Sar1-Ile8-AII binding to vascular AT1 receptors derived from both cell models with Ki values slightly lower (losartan) and higher (EXP 3174), respectively, than that of UP 269-6. 3. AII (1 microM) induced a weak and variable hyperplastic response (4 to 32% increase in cell number) in Wistar rat VSMC after 96 h. 4. AII (1 microM) induced a time-dependent increase in cell number in VSMC from SHR. UP 269-6 inhibited concentration-dependently this effect with an IC50 value of 159 +/- 58 nM. Losartan was clearly less potent and EXP 3174 showed nearly the same inhibitory potency, compared to UP 269-6. UP 269-6 (1 microM) inhibited nearly completely the action of AII. 5. AII (500 nM) caused maximal stimulation of protein synthesis in Wistar rat VSMC (117 +/- 36%). UP 269-6, losartan and EXP 3174 totally inhibited this stimulation with IC50 values of 28 +/- 6 nM, 3504 +/- 892 nM and 21 +/- 3 nM, respectively. 6. AII (50 nM) induced maximal stimulation of protein synthesis in SHR VSMC (237 +/- 67%). UP 269-6, losartan and EXP 3174 totally inhibited this stimulation with IC50 values of 16 +/- 3 nM, 282 +/- 122 nM and 3.3 +/- 1.0 nM, respectively. 7. UP 269-6 (75 mg kg-1 day-1) administered orally in the diet for 20 days induced a 38% reduction in neointimal area and a 36% reduction in neointima/media ratio associated with the intimal thickening induced by carotid artery balloon injury. 8. In conclusion, UP 269-6 was shown to be a potent antiproliferative agent both in vitro on AII-induced hyperplasia and hypertrophy of VSMC derived from normotensive and hypertensive rats, and in vivo upon intimal thickening induced by carotid artery balloon injury in the rat.

In vitro pharmacological characterization of UP 269-6, a novel nonpeptide angiotensin II receptor antagonist.

f1p4in vitro pharmacology of UP 269-6, a novel nonpeptide angiotensin II antagonist, was examined in radioligand binding and functional isolated tissue assays. UP 269-6 bound selectively to AT1 receptors as evidenced by the inhibition of specific [125I] Sar1, Ile8-AII binding in rat adrenal membranes (IC50 = 35.8 nM) and in cultured vascular smooth muscle cells (IC50 = 23.8 nM). UP 269-6 displayed a very high selectivity for the AT1 compared to the AT2 receptor subtype (IC50 > 10,000 nM). UP 269-6 inhibited the AII-induced contraction of isolated rabbit aortic strips. The pattern of AII antagonism suggested competitive antagonism at low concentrations (10(-10), 3 x 10(-10), 10(-9) M) of UP 269-6 and insurmountable antagonism at higher concentrations (3 x 10(-9), 10(-8), 3 x 10(-8) M). Based on the calculated pA2 values, UP 269-6 (9.86 +/- 0.25) was an angiotensin II receptor antagonist as potent as L-158,809 (9.82 +/- 0.37) and much more potent than losartan (7.96 +/- 0.38). UP 269-6 was devoid of affinity (IC50 > 10,000 nM) for many other receptors, ion channels and uptake sites, demonstrating its high specificity for AII receptors. Furthermore, this compound did not affect the contractile response to KCl or phenylephrine in rabbit aorta and exhibited no effect on angiotensin converting enzyme activity. These data demonstrate that UP 269-6 is a highly potent, selective and specific AT1 receptor antagonist.

Synthesis and angiotensin II receptor antagonist activity of C-linked pyrazole derivatives.

The synthesis and pharmacological activity of new nonpeptide angiotensin II (AII) receptor antagonists are presented. These 5-O-substituted and 5-C-substituted 3-alkylpyrazole derivatives represent a new series of antagonists and have led to the discovery of compounds with potent oral antihypertensive activity in a renal artery-ligated rat model. In vitro, they displayed a high affinity for rat adrenal AII receptors. In vivo structure-activity relationship study has shown the importance of the 4-[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl moiety for oral activity and the critical role of alkyl substituents at the 1- or 2-position. In the case of oral administration, 5-C derivatives were found to be, on the whole, more potent than 5-O derivatives. UP 221-78, 5-hydroxymethyl-3-n-propyl-1-(2,2,2-trifluoroethyl)-4- [[2'-(1H-tetrazol-5-yl)biphenyl-4-]methyl]-1H-pyrazole (79), displayed equivalent antihypertensive activity to the well known antagonist Losartan at 3 mg/kg p.o. in renal artery-ligated rats, with maximal decreases in mean arterial pressure of 60 and 63 mmHg for Losartan and UP 221-78, respectively.