Pazopanib-induced hyperbilirubinemia is associated with Gilbert's syndrome UGT1A1 polymorphism.

BACKGROUND: Pazopanib has shown clinical activity against multiple tumour types and is generally well tolerated. However, isolated elevations in transaminases and bilirubin have been observed. This study examined polymorphisms in molecules involved in pharmacokinetic and pharmacodynamic pathways of pazopanib and their association with hepatic dysfunction. METHODS: Twenty-eight polymorphisms in 11 genes were evaluated in pazopanib-treated renal cell carcinoma patients. An exploratory analysis was conducted in 116 patients from a phase II study; a replication study was conducted in 130 patients from a phase III study. RESULTS: No polymorphisms were associated with alanine aminotransferase elevation. The Gilbert's uridine-diphosphoglucuronate glucuronosyltransferase 1A1 (UGT1A1) TA-repeat polymorphism was significantly associated with pazopanib-induced hyperbilirubinemia in the phase II study. This association was replicated in the phase III study (P<0.01). Patients with TA6/TA6, TA6/TA7, and TA7/TA7 genotypes experienced median bilirubin increases of 0.31, 0.37, and 0.71 x upper limit of the normal range (ULN), respectively. Of the 38 patients with hyperbilirubinemia (> or = 1.5 x ULN), 32 (84%) were either TA7 homozygotes (n=18) or TA7 heterozygotes (n=14). For TA7 homozygotes, the odds ratio (95% CI) for developing hyperbilirubinemia was 13.1 (5.3-32.2) compared with other genotypes. CONCLUSIONS: The UGT1A1 polymorphism is frequently associated with pazopanib-induced hyperbilirubinemia. These data suggest that some instances of isolated hyperbilirubinemia in pazopanib-treated patients are benign manifestations of Gilbert's syndrome, thus supporting continuation of pazopanib monotherapy in this setting.

Effects of liposome dose and the presence of lymphosarcoma cells on blood clearance and tissue distribution of large unilamellar liposomes in mice.

Large unilamellar liposomes (50 to 500 mumol of lipid per kg) were injected i.v. or i.p. into normal and lymphosarcoma-bearing mice. The percentage of the dose remaining in the blood and that accumulated in liver, spleen, and various other organs was measured 4 hr after injection. The results indicate that liposomes cause a dose-dependent saturation of the hepatic and splenic clearance capacities. One day after injection of 10(6) lymphosarcoma cells, the capacity of the tumor-bearing mice to eliminate liposomes from the blood (in a 4-hr period) was inhibited 30 to 50%. This could be ascribed to a decreased activity of the reticuloendothelial system caused by the tumor cells, as was indicated by the simultaneous inhibition of carbon clearance. Six days after injection of the lymphosarcoma cells, the elimination of liposomes from the blood in tumor-bearing mice was restored to the value in normal mice. The possible involvement of tumor cells in the uptake of liposomes by the liver was investigated morphologically after i.v. injection of peroxidase-containing liposomes into lymphosarcoma-bearing mice. Liposome-entrapped peroxidase activity was never observed in the tumor cells. The results presented here indicate that the lymphosarcoma cells do not directly participate in the hepatic accumulation of liposomes, although their mere presence may have significant indirect effects on the elimination of liposomes from the blood and on their tissue distribution.

In vivo fate of large unilamellar sphingomyelin-cholesterol liposomes after intraperitoneal and intravenous injection into rats.

We investigated the fate of intraperitoneally and intravenously injected reverse phase evaporation vesicles of fairly uniform size (100-200 nm) with respect to blood clearance, tissue distribution and integrity in vivo. The vesicles are composed of sphingomyelin and cholesterol in a molar ratio 3 : 2 and contain 125I-labeled poly(vinyl pyrrolidone) in the aqueous compartment. It is shown that following an intraperitoneal injection the vesicles are transported intact, and not associated with cells, from the peritoneal cavity to the blood and are subsequently taken up mainly by liver and spleen, where, particularly in liver, the phospholipid is partially metabolized. After an intraperitoneal injection the rate of vesicle-uptake by liver and spleen is reduced by a factor of 2-3 compared to the rate of vesicle-uptake by liver and spleen following an intravenous injection. The peritoneal cavity functions as a reservoir of vesicles for some hours. The rates of blood clearance and uptake of the vesicles by liver and spleen appear to be slower than that found for vesicles of different lipid composition.

Reversible depression of the reticuloendothelial system by liposomes.

The effect of various doses of different types (reverse phase evaporation vesicles and small unilamellar vesicles) of intravenously injected liposomes on reticuloendothelial activity, as measured by the blood clearance rate of intravenously injected carbon, was investigated. Also the effect of pretreatment with reverse phase evaporation vesicles on blood clearance and tissue distribution of a second dose of similar vesicles was determined. For all concentrations used reverse phase evaporation vesicles caused a reduction in reticuloendothelial activity at least up to 4 h after injection. 24 h after administration the rate of carbon clearance returned to the control level. On the contrary small unilamellar vesicles did not block reticuloendothelial activity. Pretreatment with reverse phase evaporation vesicles (250 mumol/kg) caused an increased blood level and a decreased hepatic uptake of a second dose of the vesicles, injected 1 h after the first dose. This seems to be due to a depression of reticuloendothelial activity and not to a depletion of opsonins. Pretreatment with small unilamellar vesicles (250 mumol/kg) had no significant influence on the tissue distribution of a second dose of vesicles. Our results clearly indicate that reverse phase evaporation vesicles cause a reversible depression of reticuloendothelial activity and this depression seems to be induced by a saturation of reticuloendothelial cells with liposomes.

An architecture for the fusion site of influenza hemagglutinin.

The recent finding that more than one Influenza hemagglutinin (HA) is required at the fusion site for HA-expressing fibroblasts, together with the crystal structure of HA at neutral pH, provide the basic elements of a plausible model for this fusion site. Within an aggregate of HA trimers at low pH, we propose fusion intermediates which are based upon a minimal alteration to the known neutral pH structure of HA and which should have reasonable activation energies. This is the first model of a glycoprotein-mediated fusion site which explicitly accounts for the disposition of the lipids within these intermediates. While the fusion site created by HA will not be the same as that of eukaryotic fusion complexes, general characteristics could be shared.

ITC recommendations for transporter kinetic parameter estimation and translational modeling of transport-mediated PK and DDIs in humans.

This white paper provides a critical analysis of methods for estimating transporter kinetics and recommendations on proper parameter calculation in various experimental systems. Rational interpretation of transporter-knockout animal findings and application of static and dynamic physiologically based modeling approaches for prediction of human transporter-mediated pharmacokinetics and drug-drug interactions (DDIs) are presented. The objective is to provide appropriate guidance for the use of in vitro, in vivo, and modeling tools in translational transporter science.

Fusion of influenza virus with sialic acid-bearing target membranes.

We have monitored the fusion of intact A/PR/8/34 influenza virus with glycophorin-bearing liposomes and with ganglioside- (GD1a-) containing liposomes. The lipid bilayers of the glycophorin-bearing liposomes had several compositions, including pure dioleoylphosphatidylethanolamine (DOPE), pure egg phosphatidylethanolamine (EPE), and pure dioleoylphosphatidylcholine (DOPC). Examination of the temperature dependence of fusion for these and other compositions showed that even if the lipids are competent to form inverted hexagonal phases (HII), there is no enhancement of the fusion rate constant at the L alpha-HII phase transition temperature of the lipids, TH. Thus, the HII phase transition is not involved in the HA-mediated fusion mechanism. However, this mechanism is sensitive to lipid composition, in that PC bilayers fused more slowly than PE-containing bilayers above 20 degrees C. These results show that the HA-mediated fusion mechanism depends primarily upon specific lipid-protein interactions, although the fundamental parameters of lipid phase stability (interstice stabilization and monolayer spontaneous radius of curvature) may also be important. The fact that HII phase-component lipid bilayers in the glycophorin liposomes do not enhance the HA-mediated fusion rate strongly suggests that substantial bilayer-bilayer contact is not involved in HA-mediated fusion. Previously, we have shown that glycoprotein-bearing liposomes bind to HA-expressing cells specifically through HA-glycophorin interactions and that fusion is mediated by HAs not bound to glycophorin. Thus, with respect to the target membrane, the fusion site involves just the lipid bilayer. Our results with GD1a-containing liposomes strongly suggest that HAs bound to this sialic acid-bearing molecule are likewise incapable of participating in the fusion site. This could be due to a diminished lateral mobility of the HAs simultaneously bound to both closely apposed membranes. Finally, we find that the low-pH-induced viral inactivation is inhibited by binding to either glycophorin- or GD1a-containing target membranes.

Effect of medium-chain glycerides on physiological properties of rabbit intestinal epithelium in vitro.

Medium chain glycerides (MCGs) have been reported to enhance intestinal absorption of hydrophilic drugs. However, the mechanisms involved in absorption enhancement are not well understood. The effects of MCGs (CapMul MCM) on physiological properties of rabbit ileum and distal colon, including active ion transport, transepithelial resistance (Rt) and passive permeability, have been investigated in vitro. CapMul MCM inhibited active ion transport (measured as a decrease in short-circuit current, Isc) in both intestinal segments in a concentration-dependent manner. The inhibition of Isc was rapidly reversible (within 100 min) upon removal of CapMul MCM. The data indicate that CapMul MCM preferentially affected ion transport by villus cells in the ileum and surface cells in the distal colon. Ion transport in crypt cells in both segments was not significantly altered. Rt of the ileum was not significantly affected by 5% CapMul MCM, while mannitol transport was 6 fold enhanced. Treatment of distal colon with 1% CapMul MCM reduced Rt by 95%, while mannitol transport was 100 fold enhanced. In a parallel experiment, mucosal(m)-to-serosal(s) transport of cephalexin, a beta-lactam antibiotic, in the ileum was about 40% reduced in the presence of 5% CapMul MCM, whereas transport in the s-to-m direction was 2.5 fold enhanced. Treatment of the distal colon with 1% CapMul MCM resulted in 25 fold enhancement of cephalexin transport in either direction. These results suggest that absorption enhancement by MCGs results from an increased permeability of the intestine confined to the villus or surface epithelium.

Fusion of phosphatidylethanolamine-containing liposomes and mechanism of the L alpha-HII phase transition.

The initial kinetics of fusion and leakage of liposomes composed of N-methylated dioleoylphosphatidylethanolamine (DOPE-Me) have been correlated with the phase behavior of this lipid. Gagné et al. [Gagné, J., Stamatatos, L., Diacovo, T., Hui, S. W., Yeagle, P., & Silvius, J. (1985) Biochemistry 24, 4400-4408] have shown that this lipid is lamellar (L alpha) below 20 degrees C, is hexagonal (HII) above 70 degrees C, and shows isotropic 31P NMR resonances at intermediate temperatures. This isotropic state is also characterized by complex morphological structures. We have prepared DOPE-Me liposomes at pH 9.5 and monitored the temperature dependence of the mixing of aqueous contents, leakage, and changes in light scattering upon reduction of the pH to 4.5. At and below 20 degrees C, where the lipid is in the L alpha phase, there is very little aggregation or destabilization of the liposomes. Between 30 and 60 degrees C, i.e., where the lipid is in the isotropic state, the initial rates of liposome fusion (mixing of aqueous contents) and leakage increase. At temperatures approaching that where the hexagonal HII phase transition occurs, the initial rates and extents of fusion decrease, whereas leakage is enhanced. Similar results were found for dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine (2:1) liposomes. These results clearly establish a common mechanism between the appearance of the isotropic state (between the L alpha and HII phases) and the promotion of liposome fusion. We propose a simple model to explain both the observed behavior of phosphatidylethanolamine-containing membranes with respect to liposome fusion and/or lysis and the beginning of the L alpha-HII phase transition.

H+- and Ca2+-induced fusion and destabilization of liposomes.

A new liposome fusion assay has been developed that monitors the mixing of aqueous contents at neutral and low pH. With this assay we have investigated the ability of H+ to induce membrane destabilization and fusion. The assay involves the fluorophore 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and its quencher N,N'-p-xylylenebis(pyridinium bromide) (DPX). ANTS is encapsulated in one population of liposomes and DPX in another, and fusion results in the quenching of ANTS fluorescence. The results obtained with the ANTS/DPX assay at neutral pH give kinetics for the Ca2+-induced fusion of phosphatidylserine large unilamellar vesicles (PS LUV) that are very similar to those obtained with the Tb3+/dipicolinic acid (DPA) assay [Wilschut, J., & Papahadjopoulos, D. (1979) Nature (London) 281, 690-692]. ANTS fluorescence is relatively insensitive to pH between 7.5 and 4.0. Below pH 4.0 the assay can be used semiquantitatively by correcting for quenching of ANTS due to protonation. For PS LUV it was found that, at pH 2.0, H+ by itself causes mixing of aqueous contents, which makes H+ unique among the monovalent cations. We have shown previously that H+ causes a contact-induced leakage from liposomes composed of phosphatidylethanolamine and the charged cholesteryl ester cholesteryl hemisuccinate (CHEMS) at pH 5.0 or below, where CHEMS becomes protonated. Here we show that H+ causes lipid mixing in this pH range but not mixing of aqueous contents. This result affirms the necessity of using both aqueous space and lipid bilayer assays to comprehend the fusion event between two liposomes.

Distribution and metabolism of lipsome-encapsulated and free 1-beta-D-arabinofuranosylcytosine (Ara-C) in dog and mouse tissues.

The effect of liposome encapsulation of the metabolic activation (phosphorylation) and degradation (deamination) of arabinofuranosylcytosine (Ara-C) in liver and spleen of dogs and mice was investigated. Ara-C in free or liposome-encapsulated form was administered i.v. to dogs and DBA2/CR mice bearing leukemia L1210. At various times after injection the concentration of Ara-C and Ara-C metabolites in the blood, liver and spleen was measured. It was shown that liposome encapsulation results in an increased Ara-C/arabinofuranosyluracil ratio in the liver and spleen of dogs and leukemic mice and that encapsulated Ara-C generates a sustained level of Ara-C triphosphate in the liver and spleen of leukemic mice. These results clearly indicate that 1) encapsulated Ara-C is protected against deamination in the liver, 2) encapsulated Ara-C is slowly released from liposomes in liver and spleen and 3) that liposomes may act as a local depot for Ara-C in these tissues.

Destabilization of phosphatidylethanolamine liposomes at the hexagonal phase transition temperature.

We have examined whether there is a relationship between the lamellar-hexagonal phase transition temperature, TH, and the initial kinetics of H+- and Ca2+-induced destabilization of phosphatidylethanolamine (PE) liposomes. The liposomes were composed of dioleoylphosphatidylethanolamine, egg phosphatidylethanolamine (EPE), or phosphatidylethanolamine prepared from egg phosphatidylcholine by transesterification (TPE). These lipids have well-spaced lamellar-hexagonal phase transition temperatures (approximately 12, approximately 45, and approximately 57 degrees C) in a temperature range that allows us to measure the initial kinetics of bilayer destabilization, both below and above TH. The liposomes were prepared at pH 9.5. The TH of EPE and TPE was measured by using differential scanning calorimetry, and it was found that the TH was essentially the same at low pH or at high pH in the presence of 20 mM Ca2+. At temperatures well below TH, either at pH 4.5 or at pH 9.5 in the presence of Ca2+, the liposomes aggregate, leak, and undergo lipid mixing and mixing of contents. We show that liposome/liposome contact is involved in the destabilization of the PE liposomes. The temperature dependence of leakage, lipid mixing, and mixing of contents shows that there is a massive enhancement in the rate of leakage when the temperature approaches the TH of the particular PE and that lipid mixing appears to be enhanced. However, the fusion (mixing of aqueous contents) is diminished or even abolished at temperatures above TH. At and above the TH, a new mechanism of liposome destabilization arises, evidently dependent upon the ability of the PE molecules to adapt new morphological structures at these temperatures. We propose that this destabilization demarks the first step in the pathway to the eventual formation of the HII phase. Thus, the polymorphism accessible to PE is a powerful agent for membrane destabilization, but additional factors are required for fusion.

Destabilization of phosphatidylethanolamine-containing liposomes: hexagonal phase and asymmetric membranes.

We have measured the temperature of the L alpha-HII phase transition, TH, for several types of phosphatidylethanolamine (PE), their binary mixtures, and several PE/cholesteryl hemisuccinate (CHEMS) mixtures. We have shown for liposomes composed of pure PE and in mixtures with CHEMS that there is an aggregation-mediated destabilization which is greatly enhanced at and above TH. We now ask the question: How well can a dioleoylphosphatidylethanolamine/CHEMS liposome, for example, destabilize TPE (transesterified from egg phosphatidylcholine)/CHEMS liposome and vice versa? We use Ca2+ and H+ to induce aggregation and to provide different values of TH: the TH of the PE/CHEMS mixture is much lower at low pH than with Ca2+. We find that if the temperature is above the TH of one lipid mixture, e.g., A, and below the TH of the other lipid mixture, e.g., B, then the destabilization sequence [measured by the fluorescent 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylylenebis(pyridinium bromide) leakage assay] is AA greater than AB much greater than BB. That is, the bilayer of the lipid A (which on its own would end up in the HII phase) destabilizes itself better than it destabilizes the bilayer of lipid B (which on its own would remain in the L alpha phase). The BB contact is the least unstable. From these experiments, we conclude that the enhanced destabilization of membranes provided by the polymorphism accessible to these lipids above TH is effective even if only one of the apposed outer monolayers is HII phase competent. The surprising result is that if the temperature is above the TH of both lipid mixtures, then the destabilization sequence is AB greater than AA, BB. That is, the mixed bilayers are destabilized more by contact than either of the pure pairs. We believe that this is due to specific differences in the kinetics of aggregation or close approach of the membranes. Similar results were obtained with pure PE liposomes induced to aggregate by Ca2+ at pH 9.5. We also found that the kinetics of low-pH-induced leakage from PE/CHEMS liposomes were initially faster when the CHEMS on both sides of the bilayer is fully protonated. However, in a citrate buffer, which cannot cross intact membranes, the leakage was eventually faster. Flip-flop of the protonated CHEMS to the inner monolayer can explain this observation.

pH-induced destabilization of phosphatidylethanolamine-containing liposomes: role of bilayer contact.

The mechanism of pH-induced destabilization of liposomes composed of phosphatidylethanolamine and a charged cholesteryl ester was studied by following the release of encapsulated aqueous contents. The kinetics of release were measured continuously by using the water-soluble fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid in combination with the water-soluble quencher p- xylylenebis (pyridinium) bromide. With this fluorescence assay, release of contents from liposomes composed of phosphatidylethanolamine and cholesteryl hemisuccinate was shown to be a function of pH, ratio of phosphatidylethanolamine to cholesteryl hemisuccinate, and acyl chain composition of the phosphatidylethanolamine. Leakage was very slow at pH 5.5 and increased dramatically with decreasing pH down to 4.0. Replacing phosphatidylethanolamine by phosphatidylcholine eliminated the effect of pH on leakage. Analysis of the kinetics of release by a mass action model demonstrated that bilayer destabilization and leakage occur subsequent to aggregation. The requirement of bilayer contact for destabilization has been found previously for acidic phospholipid bilayers in the presence of divalent cation and for saturated phosphatidylcholine bilayers below the isothermal phase transition temperature. The phosphatidylethanolamine-containing bilayers examined here satisfy the same requirement.

Physiological considerations in the design of particulate dosage forms for oral vaccine delivery.

The gastrointestinal tract provides a variety of morphological (e.g. epithelial cells, mucus) and physiological (e.g. enzymes, pH, transporters) barriers to the absorption of peptides and proteins. Approaches to overcome these barriers have included the use of particulates which are taken up by specialized mechanisms present in M cells of the gastrointestinal tract. Due to its limited capacity, this approach has found particular application in the delivery of vaccines. In this review, morphological and physiological characteristics of the gastrointestinal tract which influence the design of particulates for oral delivery will be presented. Particulates have been designed to resist luminal factors responsible for limiting absorption and to target a specialized cell population, the M cells, within the gastrointestinal tract employing both physical and biological approaches (e.g. charge, size, hydrophobicity, surface ligands such as lectins). For vaccines, this approach may have 'particular' attraction due to the signal magnification which can be accomplished in the gut associated lymphoid tissue (GALT). Recent studies have demonstrated that epithelial cells can be converted to M cells following exposure to Peyer's patch lymphocytes. Future studies designed to identify the factor(s) responsible for transient conversion of epithelial cells to M cells could provide an approach to enhance efficiency of vaccine delivery.

Size-dependent permeability of hydrophilic probes across rabbit colonic epithelium.

Colon-specific delivery of metabolically labile molecules, such as proteins and peptides, is of particular interest in pharmaceutical research. Among the factors that may influence the permeability of drug molecules across colonic mucosa are their molecular weight and geometry. The purpose of this study was to evaluate the influence of molecular geometry on in vitro permeability across rabbit distal colonic epithelia. Permeability of radiolabeled hydrophilic probes with different molecular weights and geometries across isolated rabbit distal colonic tissue was evaluated by means of the Ussing chamber technique. The hydrodynamic radii of the probes (an indicator of molecular geometry) were estimated by theoretical models as well as dynamic light scattering. We conducted the permeability studies in the presence and absence of the epithelial cells to evaluate the contribution of the underlying connective tissue to the overall in vitro permeability across the colonic mucosa. The rank order of the permeability of the markers was mannitol > lactulose > polyethylene glycol (PEG) 400 > PEG 900 > PEG 4000, which is consistent with their molecular weights and estimated hydrodynamic radii. The permeability of inulin, a polyfructose molecule with a molecular weight of about 5000, however, was approximately the same as that of PEG 900 (molecular weight about 900). When the epithelial cells were removed, for the homologous series of PEGs, the permeabilities were proportional to their free diffusion coefficients in water. It appears that for the PEG and lactulose probes, theoretical estimation of the hydrodynamic radii, which assumes the molecules to be spherical in shape, provides a good basis for the dependence of permeability on geometry. The relatively high permeability of inulin seems to be due to its compact structure. The PEG permeability values in the absence of epithelial cells, in combination with their diffusion coefficients, indicate that the underlying connective tissue does not contribute to the overall permeability of these molecules across colonic mucosa in vitro.

On the correlation between HII phase and the contact-induced destabilization of phosphatidylethanolamine-containing membranes.

The abundance of phosphatidylethanolamine (PtdEtn) in biological membranes and the capacity of this lipid to sustain nonbilayer structures have been promoted as evidence for a role of PtdEtn in biological fusion processes. To date there has been no direct evidence of a connection between the kinetics of bilayer destabilization and the polymorphism accessible to PtdEtn. We have developed a model system to examine this point directly using the proton-induced destabilization of PtdEtn/cholesterylhemisuccinate unilamellar liposomes. We find that the initial rate of bilayer mixing rapidly increases with temperature and reaches a maximal level just below the HII-phase transition temperature. The leakage from these liposomes rapidly increases, both in rate and extent, within the HII-phase transition temperature range. Of an even greater significance is that at no temperature is there any mixing of aqueous contents within the liposomes. Thus, these lipids can begin to undergo the lamellar- to HII-phase transition at the stage of two apposed liposomes. However, the nonbilayer structures formed do not cause fusion--i.e., the concomitant mixing of aqueous contents.