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.

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.

Prediction of the intestinal absorption of endothelin receptor antagonists using three theoretical methods of increasing complexity.

PURPOSE: Three new computational strategies have been evaluated for their ability to predict intestinal membrane permeability to a series of endothelin receptor antagonists. METHODS: The three methods were evaluated using a set of ten nonpeptide endothelin receptor antagonists. The simplest method, "the rule of five", is based on 2D parameters such as the number of potential hydrogen bonds, molecular weight and calculated lipophilicity. A method based on molecular mechanics calculations is used to calculate 3D parameters such as polar and non-polar parts of the molecular surface area. The third method uses quantum mechanics to calculate molecular properties related to the valence region. RESULTS: Descriptors derived by the latter two methods correlated well with permeability coefficients of the endothelin receptor antagonists. On the other hand, the rule of five failed to discriminate between drugs with high and low permeability. CONCLUSIONS: Molecular surface descriptors and descriptors derived from quantum mechanics are potentially useful for the virtual screening of the permeability of the intestinal membrane to endothelin receptor antagonists.

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.

Nonpeptide endothelin receptor antagonists. VI: Pharmacological characterization of SB 217242, a potent and highly bioavailable endothelin receptor antagonist.

This study describes the pharmacological characterization of SB 217242, a highly potent orally bioavailable nonpeptide antagonist of both endothelin type A (ETA) and endothelin type B (ETB) receptors. In human cloned ETA and ETB receptors, SB 217242 produced concentration-dependent displacement of [125]I-endothelin-1 (ET-1) in both receptor subtypes with Ki values of 1.1 and 111 nM, respectively. SB 217242 produced concentration-dependent, parallel rightward shifts in the ET-1 concentration-response curves in rat isolated aorta and human isolated pulmonary artery (ETA receptor-mediated vascular contraction) with Kb values of 4.4 and 5.0 nM, respectively. SB 217242 was 4-, 62- and 125-fold more potent as an ETA receptor antagonist than the previously reported compounds BQ-123, PD 142893 and Ro 46-2005, respectively. SB 217242 (10 microM) did not produce significant effects against contraction produced by other vasoactive agents. SB 217242 produced concentration-dependent antagonism of responses produced by ETB receptor activation as demonstrated by antagonism of sarafotoxin S6c-mediated contraction in the rabbit isolated pulmonary artery with a Kb value of 352 nM. In vitro cell monolayers of Caco-2 cells had high permeability to SB 217242. In vivo pharmacokinetics in the rat confirmed that SB 217242 was rapidly absorbed from the gastrointestinal tract with a bioavailability of 66%. The SB 217242 plasma half-life in rats after intraduodenal administration was 3.3 hr, with a systemic clearance of 27.3 ml/min/kg. Orally administered SB 217242 (0.3-30 mg/kg) produced dose-dependent inhibition of the pressor response to exogenous ET-1 in conscious rats; with a dose of 30 mg/kg p.o., inhibition was observed for at least 5.5 hr. The present study demonstrates that SB 217242 is a highly potent antagonist of both ETA and ETB receptors. In addition, SB 217242 has high in vitro permeability and high oral bioavailability. SB 217242 represents a new orally active pharmacological tool that should assist in the elucidation of the chronic role of endothelin in pathophysiology.

Water-in-oil microemulsions containing medium-chain fatty acids/salts: formulation and intestinal absorption enhancement evaluation.

PURPOSE: Water-in-oil (w/o) microemulsions have been developed which, in addition to non-ionic medium-chain glycerides, incorporate ionic lipids, primarily medium-chain fatty acids, such as caprylic (C8) capric (C10) and lauric (C12) acids and their corresponding sodium salts. The absorption enhancing activity of w/o microemulsions incorporating these lipids was evaluated in the rat using Calcein (MW = 623) a water-soluble and poorly absorbed marker molecule. METHODS: Phase diagrams were constructed where C8/C10 or C12 fatty acids were treated as lipophilic surfactants and their sodium salts as hydrophilic ones. The anesthetised rat model was employed to evaluate Calcein absorption upon a single intraduodenal administration from a solution and the various w/o microemulsions. RESULTS: A wide range of clear and transparent w/o microemulsions were obtained at ambient temperature either in liquid or solid form when a fixed blend of medium chain fatty acid/salt was titrated by a fixed ratio of the oil containing the oil-soluble mono- and diglycerides and deionized water or physiological saline. Upon intraduodenal administration in the anesthetised rat, the absorption of Calcein was improved from about 2% in aqueous solution up to about 37% in w/o microemulsions. Solid and liquid formulations were equally effective in improving bioavailability. The absorption enhancement activity of the fatty acids/salts followed the order C8 approximately C10 > C12. Absorption enhancement of Calcein was significantly reduced in the absence or presence of low levels of C8/C10 mono-/diglycerides. CONCLUSIONS: These results further support the use of medium-chain glycerides and fatty acids/salts in microemulsion formulations to improve intestinal absorption of water-soluble compounds.

Formulation and intestinal absorption enhancement evaluation of water-in-oil microemulsions incorporating medium-chain glycerides.

We developed self-emulsifying water-in-oil (w/o) microemulsions incorporating medium-chain glycerides and measured their conductance, viscosity, refractive index and particle size. Formulation of Calcein (a water-soluble marker molecule, MW = 623), or SK&F 106760 (a water-soluble RGD peptide, MW = 634) in a w/o microemulsion having a composition of Captex 355/Capmul MCM/Tween 80/Aqueous (65/22/10/3, % w/w), resulted in significant bioavailability enhancement in rats relative to their aqueous formulations. Upon intraduodenal administration the bioavailability was enhanced from 2% for Calcein in isotonic Tris, pH 7.4 to 45% in the microemulsion and from 0.5% for SK&F 106760 in physiological saline to 27% in the microemulsion formulation. The microemulsion did not induce gross changes in GI mucosa at a dosing volume of 3.3 ml/kg. These results suggest that water-in-oil microemulsion systems may be utilized for enhancement of intestinal drug absorption.

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 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.

Binding of soluble CD4 proteins to human immunodeficiency virus type 1 and infected cells induces release of envelope glycoprotein gp120.

Human immunodeficiency virus (HIV) infects cells after binding of the viral envelope glycoprotein gp120 to the cell surface recognition marker CD4. gp120 is noncovalently associated with the HIV transmembrane envelope glycoprotein gp41, and this complex is believed responsible for the initial stages of HIV infection and cytopathic events in infected cells. Soluble constructs of CD4 that contain the gp120 binding site inhibit HIV infection in vitro. This is believed to occur by competitive inhibition of viral binding to cellular CD4. Here we suggest an alternative mechanism of viral inhibition by soluble CD4 proteins. We demonstrate biochemically and morphologically that following binding, the soluble CD4 proteins sT4, V1V2,DT, and V1[106] (amino acids 1-369, 1-183, and -2 to 106 of mature CD4) induced the release of gp120 from HIV-1 and HIV-1-infected cells. gp120 release was concentration-, time-, and temperature-dependent. The reaction was biphasic at 37 degrees C and did not take place at 4 degrees C, indicating that binding of soluble CD4 was not sufficient to release gp120. The appearance of free gp120 in the medium after incubation with sT4 correlated with a decrease in envelope glycoprotein spikes on virions and exposure of a previously cryptic epitope near the amino terminus of gp41 on virions and infected cells. The concentration of soluble CD4 proteins needed to induce the release of gp120 from virally infected cells also correlated with those required to inhibit HIV-mediated syncytium formation. These results suggest that soluble CD4 constructs may inactivate HIV by inducing the release of gp120. We propose that HIV envelope-mediated fusion is initiated following rearrangement and/or dissociation of gp120 from the gp120-gp41 complex upon binding to cellular CD4, thus exposing the fusion domain of gp41.

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.

Fusion of influenza hemagglutinin-expressing fibroblasts with glycophorin-bearing liposomes: role of hemagglutinin surface density.

Influenza virus gains access to the cytoplasm of its host cell by means of a fusion event between viral and host cell membrane. Fusion is mediated by the envelope glycoprotein hemagglutinin (HA) and is triggered by low pH. To learn how many hemagglutinin trimers are necessary to cause membrane fusion, we have used two NIH 3T3 fibroblast cell lines that express HA protein at different surface densities. On the basis of quantitations of the number of HA trimers per cell and the relative surface areas of the two cell lines, the HAb-2 cells have a 1.9-fold higher plasma membrane surface density than the GP4F cells. The membrane lateral diffusion coefficient and the mobile fraction for HA is the same for both cell lines. A Scatchard analysis of the binding of glycophorin-bearing liposomes to the cells showed 1700 binding sites for the GP4F cells and 3750 binding sites for the HAb-2 cells, with effectively the same liposome-cell binding constant, about 7 x 10(10) M-1. Binding was specific for glycophorin on the liposomes and HA expressed on the cells. A competition experiment employing toxin-containing and empty liposomes allowed us to quantitate the number of liposomes that fused per cell, which was a small constant fraction of the number of bound liposomes. For the HAb-2 cells, about 1 in every 70 bound liposomes fused and for the GP4F cells about 1 in every 300 bound liposomes fused. Hence, the HAb-2 cells showed 4.4 times more fusion per bound liposome, even though the surface density of HA was only 1.9 times greater. We conclude the following: (i) One HA trimer is not sufficient to induce fusion. (ii) The HA bound to glycophorin is not the HA that induces fusion. That is, even though each HA has a binding and a fusion function, those functions are not performed by the same HA trimer.

Membrane fusion and inverted phases.

We have found a correlation between liposome fusion kinetics and lipid phase behavior for several inverted phase forming lipids. N-Methylated dioleoylphosphatidylethanolamine (DOPE-Me), or mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), will form an inverted hexagonal phase (HII) at high temperatures (above TH), a lamellar phase (L alpha) at low temperatures, and an isotropic/inverted cubic phase at intermediate temperatures, which is defined by the appearance of narrow isotropic 31P NMR resonances. The phase behavior has been verified by using high-sensitivity DSC, 31P NMR, freeze-fracture electron microscopy, and X-ray diffraction. The temperature range over which the narrow isotropic resonances occur is defined as delta TI, and the range ends at TH. Extruded liposomes (approximately 0.2 microns in diameter) composed of these lipids show fusion and leakage kinetics which are strongly correlated with the temperatures of these phase transitions. At temperatures below delta TI, where the lipid phase is L alpha, there is little or no fusion, i.e., mixing of aqueous contents, or leakage. However, as the temperature reaches delta TI, there is a rapid increase in both fusion and leakage rates. At temperatures above TH, the liposomes show aggregation-dependent lysis, as the rapid formation of HII phase precursors disrupts the membranes. We show that the correspondence between the fusion and leakage kinetics and the observed phase behavior is easily rationalized in terms of a recent kinetic theory of L alpha/inverted phase transitions. In particular, it is likely that membrane fusion and the L alpha/inverted cubic phase transition proceed via a common set of intermembrane intermediates.

Physiological levels of diacylglycerols in phospholipid membranes induce membrane fusion and stabilize inverted phases.

In the preceding paper (Ellens et al., 1989), it was shown that liposome fusion rates are substantially enhanced under the same conditions which induce isotropic 31P NMR resonances in multilamellar dispersions of the same lipid. Both of these phenomena occur within the same temperature interval, delta TI, below the L alpha/HII phase transition temperature, TH. TH and delta TI can be extremely sensitive to the lipid composition. The present work shows that 2 mol% of diacylglycerols like those produced by the phosphatidylinositol cycle in vivo can lower TH, delta TI, and the temperature for fast membrane fusion by 15-20 degrees C. N-Monomethylated dioleoylphosphatidylethanolamine is used as a model system. These results show that physiological levels of diacylglycerols can substantially increase the susceptibility of phospholipid membranes to fusion. This suggests that, in addition to their role in protein kinase C activation, diacylglycerols could play a more direct role in the fusion event during stimulus-exocytosis coupling in vivo.