Pharmacokinetic and imaging studies in patients receiving a formulation of liposome-associated adriamycin.

Pharmacokinetic and imaging studies in 19 patients receiving liposome-entrapped adriamycin (L-ADM) were carried out within the framework of a Phase I clinical trial (Gabizon et al., 1989a). The formulation of L-ADM tested consisted of 0.2 microM-extruded multilamellar vesicles composed of egg phosphatidylcholine, egg-derived phosphatidyl-glycerol (PG), cholesterol, and ADM intercalated in the fluid lipid bilayer. Plasma clearance of total drug extracted from the plasma after L-ADM infusion followed a biexponential curve with a pattern similar to that reported for free ADM. The plasma concentration of drug circulating in liposome-associated from was also measured in a subgroup of seven patients. Liposome-associated drug was found to be rapidly cleared from plasma. Its ratio to non-liposome-associated drug appeared to correlate with liver reserve, with highest ratios in patients with normal liver function. Liposome clearance, as measured by the plasma concentration of PG in three patients was slower than the clearance of liposome-associated ADM, suggesting that liposomes lose part of their drug payload during circulation. To learn about the liposome organ distribution, imaging studies were carried out with 111Indium-deferoxamine labelled liposomes of the same composition. Liposomes were cleared predominantly by liver and spleen and to a lesser extent by bone marrow in seven out of nine patients. In two patients with active hepatitis and severe liver dysfunction, there was minimal liver uptake and increased spleen and bone marrow uptake. Except for one hepatoma patient, intrahepatic and extrahepatic tumours were not imaged by liposomes, suggesting that liposome uptake is restricted to cells of the reticulo-endothelial system (RES).(ABSTRACT TRUNCATED AT 250 WORDS)

Volume regulation of nerve terminals.

Pinched-off presynaptic nerve terminals (synaptosomes) possess significant regulatory volume increase (RVI) and regulatory volume decrease (RVD) capabilities. Following a swelling induced by a hypotonic challenge, the synaptosomes regulate their volume and adjust it, in 2 min, to within 5% of its initial value (RVD) at an initial rate of -0.77 +/- 0.10%/s (mean +/- SEM). Following a shrinking induced by a hypertonic challenge, the synaptosomes also regulate their volume at an initial rate of 0.18 +/- 0.02%/s (RVI), resulting in a new steady state, reached within 5-10 min, with a synaptosomal volume below the original volume. The omission of Na+ or K+ ions from the extrasynaptosomal medium reduces the initial rate of RVI by 72.5 and 66.5%, respectively. The "loop diuretics" bumetanide and furosemide significantly inhibited the RVI of the synaptosomes. In contrast, ouabain, amiloride, or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid did not have any significant effect on RVI parameters. Furthermore, bumetanide-sensitive 86Rb uptake by rat brain synaptosomes was stimulated threefold by a hypertonic perturbation of 30%. Thus we conclude that the RVI of synaptosomes is mainly due to a stimulation of the Na+, K+, Cl- co-transport system induced by the synaptosomal shrinking following the hypertonic challenge.

Basic characterization of an ouabain-resistant, bumetanide-sensitive K+ carrier-mediated transport system in J774.2 mouse macrophage-like cell line and in variants deficient in adenylate cyclase and cAMP-dependent protein kinase activities.

86Rb(K+) transport across the plasma membrane of macrophage-like cells was studied. The cells used were the wild-type J774.2 and its two variants, CT2 cells, deficient in adenylate cyclase, and J7H1 cells, deficient in cAMP-dependent protein kinase. In the three cell lines about 15% of the total 86Rb(K+) influx is transported by the K+ carrier-mediated transport system. The 86Rb(K+) efflux carried by the same transporter is negligible when measured in the absence of ouabain in the medium. Therefore this carrier conducts a net inward flux of K+ under the experimental conditions used. The transporter is sensitive to extracellular Na+ and inhibited by 'loop' diuretics; bumetanide inhibits ouabain-resistant 86Rb(K+) influx with IC50 of 0.1, 5.0, and 0.05 microM for J774.2, CT2 and J7H1 macrophages, respectively. The membrane potential of the three cells was measured, using the distribution of [3H]tetraphenylphosphonium [( 3H]TPP+) across the plasma membrane, and found to be -80.1, -108.5 and -105.1 mV for J774.2, CT2 and J7H1 cells, respectively. The addition of bumetanide to the cell medium does not alter [3H]TPP+ uptake indicating that the transporter is electrically silent. It is concluded that despite the differences in cAMP metabolism by the three macrophages, the basic characteristics of K+ carrier-mediated transport system of the three cells are very similar.

Rb+ influxes differentiate between growth arrest of cells by different agents.

The effect of cell cycle on Rb+ (K+) fluxes was studied in NIH 3T3 mouse fibroblasts. Serum starvation or isoleucine deprivation resulted in cell arrest at an early G1/G0 phase, accompanied by a marked decrease in both ouabain-sensitive and ouabain-resistant Rb+ influx. On the other hand, cells arrested at late G1/G0 phase by hydroxyurea treatment have high ouabain-sensitive and ouabain-resistant Rb+ influx. Butyric acid treatment resulted in cell arrest at an early G1/G0 phase, but in contrast to serum or isoleucine starvation did not decrease Rb+ influxes. It is thus shown that quiescent cells may have Rb+ influx rates as high as that of logarithmically growing cells. The results are consistent with the hypothesis that an increased ion permeability of the cell is initiated at a critical stage in G1/G0 phase, and that butyric acid may arrest the cell beyond that stage.

Differentiation between serum stimulation of ouabain-resistant and sensitive Rb influx in quiescent NIH 3T3 cells.

The addition of serum to quiescent NIH 3T3 mouse cell cultures resulted in a 10- to 20-fold increase of Rb influx which was resistant to ouabain, and only a three- to fourfold activation of ouabain-sensitive Rb influx. Stimulation of the ouabain-resistant Rb influx following serum addition reached its maximum within 2 min. The stimulation of ouabain-resistant Rb influx was a result of Vm increase while the Km for Rb was unchanged. Ouabain-resistant Rb influx, after serum addition, was resistant to amiloride and sensitive to ethacrynic acid. Replacing chloride in the medium by NO3-, CO3- and CH3COO- resulted in a drastic decrease in the ouabain-resistant Rb influx. It appeared, therefore, that the ouabain-resistant Rb influx in NIH 3T3 cells was Cl--dependent.