A noninternalized nondesensitized truncated AT1A receptor transduces an amplified ANG II signal.

The structural determinants of the rat angiotensin (ANG) II AT1A receptor involved in receptor internalization, desensitization, and activation are investigated by producing six mutants that had progressively larger deletions of the cytoplasmic tail (-13, -19, -24, -31, -46, and -56 residues, respectively). After stable transfection of the cDNAs into Chinese hamster ovary cells, all mutants, except the most truncated, exhibit normal [Sar1]ANG II affinities [dissociation constant (Kd) = 0.19-0.70 nM] compared with the wild-type (WT) receptor (Kd = 0.62 nM) and are able to activate a Gq/11 protein and a phospholipase C as measured by the ANG II-induced inositol phosphate (IP) turnover in the different clones. However, one of these mutants, delta 329 (deletion of 31 residues), exhibits a peculiar phenotype. This mutant shows a reduced ligand-induced internalization as measured by the acid-washing procedure (only 32% of receptors are internalized vs. 83% for WT). Moreover, the delta 329 mutant is less desensitized by a pretreatment with either ANG II (15% desensitization of ANG II-stimulated IP turnover vs. 60% for WT receptor) or the phorbol ester phorbol 12-myristate 13-acetate (no desensitization vs. 29% for WT receptor). These functional modifications of the delta 329 mutant are associated with the transduction of an amplified signal as demonstrated on both IP turnover and an integrated physiological effect of ANG II. Taken together, these data indicate that the sequence 329SLSTKMS335 of the rat AT1A receptor is involved in both receptor internalization and desensitization. This is the first demonstration that a desensitization- and internalization-defective AT1A receptor mutant is also hyperreactive and mediates augmented cellular responses.

Treatment of human prostate tumors PC-3 and TSU-PR1 with standard and investigational agents in SCID mice.

Both the PC-3 and the TSU-PR1 prostate tumor models were found to be satisfactory for chemotherapeutic investigations in ICR-SCID mice. The 30 to 60 mg fragments implanted took in all mice (as judged by 100% takes in the controls of all experiments as well as the passage mice). The tumor volume doubling time was 4.0 days for PC-3 and 2.5 days for TSU-PR1. Nine agents were evaluated IV against early stage subcutaneous PC-3 tumors, with Nano-piposulfan being the only agent highly active (4.9 log kill). Three other agents were moderately active: Taxol (1.5 log kill), Cryptophycin-8 (1.6 log kill), Vinblastine (1.0 log kill). Five agents were inactive: VP-16, Adriamycin, CisDDPt, 5-FUra, and Cyclophosphamide. Ten agents were evaluated IV against early stage subcutaneous TSU-PR1 tumors. Three agents were highly active, producing > 6 log kill and cures: Taxol (5/5 cures), Cryptophycin-8 (5/5 cures), Vinblastine (2/4 cures). Two other agents were moderately active: Nano-piposulfan (1.2 log kill), and Cyclophosphamide (1.1 log kill). Five agents were inactive: VP-16, Adriamycin, CisDDPt, 5-FUra, and BCNU. In part, activity was determined by the ability of the SCID mice to tolerate meaningful dosages of the agents. Agents producing granulocyte toxicity (e.g., Adriamycin) were poorly tolerated and appeared less active than expected. Vinblastine, producing little or no granulocyte toxicity was very well tolerated and appeared to be more active than expected.

Formulation and antitumor activity evaluation of nanocrystalline suspensions of poorly soluble anticancer drugs.

PURPOSE: Determine if wet milling technology could be used to formulate water insoluble antitumor agents as stabilized nanocrystalline drug suspensions that retain biological effectiveness following intravenous injection. METHODS: The versatility of the approach is demonstrated by evaluation of four poorly water soluble chemotherapeutic agents that exhibit diverse chemistries and mechanisms of action. The compounds selected were: piposulfan (alkylating agent), etoposide (topoisomerase II inhibitor), camptothecin (topoisomerase I inhibitor) and paclitaxel (antimitotic agent). The agents were wet milled as a 2% w/v solids suspension containing 1% w/v surfactant stabilizer using a low energy ball mill. The size, physical stability and efficacy of the nanocrystalline suspensions were evaluated. RESULTS: The data show the feasibility of formulating poorly water soluble anticancer agents as physically stable aqueous nanocrystalline suspensions. The suspensions are physically stable and efficacious following intravenous injection. CONCLUSIONS: Wet milling technology is a feasible approach for formulating poorly water soluble chemotherapeutic agents that may offer a number of advantages over a more classical approach.

Direct suppression of phagocytosis by amphipathic polymeric surfactants.

Recent studies have demonstrated that phagocytosis of colloidal particles by the mononuclear phagocytes of the liver and spleen can be controlled by either coating or stabilizing particulate carriers with the amphipathic polymeric surfactants, F108 and T908. These surfactants consist of copolymers of polypropylene oxide (PPO) and polyethylene oxide (PEO) and, when adsorbed to particulate surfaces, significantly decrease sequestration of particulates by the mononuclear phagocytes (MPS) of the liver. To evaluate these observations further, murine peritoneal macrophages were incubated for varying periods with surfactant-coated and noncoated polystyrene particles (PSPs). Phagocytosis was monitored using gamma counting and quantitative fluorescence microscopy. The data show that phagocytosis is decreased when PSPs are coated with F108 and T908. In addition, suppression of phagocytic activity was observed when cells were pretreated with the surfactant and then challenged with noncoated particles. The data confirm previous observations that polymeric surfactants consisting of PEO and PPO protect particulate carriers from rapid uptake by the MPS of the liver. Further, F108 and T908 suppress phagocytosis directly without affecting the integrity, viability, or functional state of the cell.