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.

Preclinical evaluation of manganese carbonate particles for magnetic resonance imaging of the liver.

RATIONALE AND OBJECTIVES: We characterized the physical, biological, and imaging properties of a manganese (Mn) carbonate particle suspension, a contrast agent for hepatic magnetic resonance (MR) imaging. METHODS: Mn carbonate suspensions were produced by controlled precipitation and characterized using light microscopy, transmission electron microscopy, and in vitro relaxivity studies. Efficacy of the agent was studied in normal and tumor-bearing rats using T1-weighted MR imaging. RESULTS: Following intravenous injection of Mn carbonate particles at doses ranging from 10 to 100 mumol Mn/kg, peak hepatic contrast enhancement of approximately 35% occurred from about 125 min until the termination of the MR imaging studies that varied from 125 to 305 min. Lesion conspicuity was increased because of relative intensity differences between normal liver and tumor. Data also showed that Mn carbonate particles dissolved on delivery to the liver, allowing Mn to interact with intrahepatic macromolecular complexes to provide positive contrast enhancement. CONCLUSION: Mn carbonate particles produce significant and sustained hepatic enhancement and should improve detection of small or isointense liver lesions.

Developmental pattern of ventricular atrial natriuretic peptide (ANP) expression in chronically hypoxic rats as an indicator of the hypertrophic process.

Atrial natriuretic peptide (ANP) is a natriuretic, diuretic and vasodilatory peptide normally synthesized and secreted by the atria of the adult mammalian heart. Synthesis of ANP in the ventricle has also been demonstrated in the fetus and neonate. In the adult, ventricular ANP is expressed under pathological conditions such as hypertension and congestive heart failure. The purpose of the present study was to analyse the spatial and temporal development of ANP expression in the right ventricle of the rat heart during the onset, establishment, and recovery from hypoxia-induced pulmonary hypertension and right ventricular hypertrophy (RVH). Significant RVH and immunoreactive ANP (ir-ANP) were detected in the right ventricles of hypoxic rats after only 3 days of exposure and continued to increase with the duration of hypoxia through 21 days. The presence of ir-ANP became apparent in the left ventricle as well as the right after 14 days of hypoxic exposure. Twenty-one days of normoxia following 21 days of hypoxia reduced RVH and ir-ANP to the levels seen at 3 days. Light microscopic immunohistochemistry demonstrated initial focal concentrations of ir-ANP in cardiomyocytes near the junction of the right ventricular free wall and the septum, as well as surrounding isolated blood vessels in the right ventricular wall, after 3 days of exposure. With increasing duration of hypoxic exposure, these immunoreactive areas enlarged to encompass the entire right ventricular wall and right half of the septum by 14 days. While many right ventricular cardiomyocytes were intensely stained at the light level, electron microscopic immunocytochemistry revealed only a sparse number of ANP-positive secretory granules. In immunohistochemical studies with an anti-clathrin antibody, there was a homogeneous staining pattern for clathrin in cardiomyocytes from the hypertrophied right ventricles. This pattern was not typical of the staining observed in other secretory cells which typically exhibit a perinuclear localization of clathrin. The alterations in ultrastructural immunocytochemistry for ANP suggest that ventricular ANP synthesis differs from atrial synthesis of this peptide. The differences in clathrin staining indicate that its expression may also be related to the hypertrophic adaptation of ventricular cardiomyocytes. Our results suggest that ventricular ANP expression in the adult rat is a dynamic event which is regulated by stress in the ventricular wall. The initial sites of ventricular ANP expression may represent zones of maximum tension in the ventricular wall following increased workload. To our knowledge this is the first study to demonstrate topographical changes in ventricular ANP expression in response to the development and reversal of cardiac hypertrophy.(ABSTRACT TRUNCATED AT 400 WORDS)

A clathrin-coated vesicle-mediated pathway in atrial natriuretic peptide (ANP) secretion.

Atrial Natriuretic Peptide (ANP), produced by atrial cardiomyocytes, is an endogenous hypotensive agent that brings about vasodilation and diuresis. Similar to other polypeptide hormones, ANP is synthesized as a precursor, preproANP. The preprohormone is processed intracellularly and is stored in secretory granules as the prohormone. During the events of exocytosis, the prohormone is converted to its active form, ANP. In this study, a double-label immunocytochemistry experiment was performed using ANP and clathrin antibodies to determine if the transport of this hormone is mediated by clathrin-coated vesicles. Additionally, we have isolated clathrin-coated vesicles (CVs) from adult rat atria using immunoadsorption, and have characterized the fraction by using SDS PAGE, TEM, and Western blot analysis. The data demonstrate that: (1) ANP and clathrin co-localize in myocardial tissue, (2) clathrin-coated vesicles can be isolated from adult rat atria, and (3) clathrin-coated vesicles isolated from adult atrial myocardium contain predominantly proANP. The presence of proANP in clathrin-coated vesicles suggests that this polypeptide hormone is transported intracellularly via a clathrin-mediated pathway and during transit the prohormone is not significantly converted to its active form.

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.