What is the mechanism behind increased permeation rate of a poorly soluble drug from aqueous dispersions of an amorphous solid dispersion?

Our aim was to explore the influence of micelles and microparticles emerging in aqueous dispersions of amorphous solid dispersions (ASDs) on molecular/apparent solubility and Caco-2 permeation. The ASD, prepared by hot-melt extrusion, contained the poorly soluble model drug ABT-102, a hydrophilic polymer, and three surfactants. Aqueous dispersions of the ASD were investigated at two concentrations, one above and one close to the critical micelle concentration of the surfactants blend in the extrudate. Micelles were detected at the higher concentration and no micelles at the lower concentration. Apparent solubility of ABT-102 was 20-fold higher in concentrated than in diluted dispersions, because of micelles. In contrast, Caco-2 permeation of ABT-102 was independent of the ASD concentration, but three times faster than that of crystalline suspensions. Molecular solubility of ABT-102 (equilibrium dialysis) was also independent of the ASD concentration, but by a factor 2 higher than crystalline ABT-102. The total amount of ABT-102 accumulated in the acceptor during Caco-2 experiments exceeded the initial amount of molecularly dissolved drug in the donor. This may indicate that dissolution of amorphous microparticles present in aqueous dispersions induces lasting supersaturation maintaining enhanced permeation. The hypothesis is supported by a slower drug permeation when the microparticles were removed.

The amorphous solid dispersion of the poorly soluble ABT-102 forms nano/microparticulate structures in aqueous medium: impact on solubility.

Amorphous solid dispersions (ASDs) are a promising formulation approach for poorly soluble active pharmaceutical ingredients (APIs), because they ideally enhance both dissolution rate and solubility. However, the mechanism behind this is not understood in detail. In the present study, we investigated the supramolecular and the nano/microparticulate structures that emerge spontaneously upon dispersion of an ASD in aqueous medium and elucidated their influence on solubility. The ASD, prepared by hot melt extrusion, contained the poorly soluble ABT-102 (solubility in buffer, 0.05 µg/mL), a hydrophilic polymer, and three surfactants. The apparent solubility of ABT-102 from the ASD-formulation was enhanced up to 200 times in comparison to crystalline ABT-102. At the same time, the molecular solubility, as assessed by inverse equilibrium dialysis, was enhanced two times. Asymmetrical flow field-flow fractionation in combination with a multiangle light-scattering detector, an ultraviolet detector, and a refractometer enabled us to separate and identify the various supramolecular assemblies that were present in the aqueous dispersions of the API-free ASD (placebo) and of binary/ternary blends of the ingredients. Thus, the supramolecular assemblies with a molar mass between 20,000 and 90,000 could be assigned to the polyvinylpyrrolidone/vinyl acetate 64, while two other kinds of assemblies were assigned to different surfactant assemblies (micelles). The amount of ABT-102 remaining associated with each of the assemblies upon fractionation was quantified offline with high-performance liquid chromatography-ultraviolet-visible. The polymeric and the micellar fraction contributed to the substantial increase in apparent solubility of ABT-102. Furthermore, a microparticulate fraction was isolated by centrifugation and analyzed by scanning electron microscopy, X-ray scattering, and infrared spectroscopy. The microparticles were found to be amorphous and to contain two of the surfactants besides ABT-102 as the main component. The amorphous microparticles are assumed to be the origin of the observed increase in molecular solubility ("true" supersaturation).

Amorphous solid dispersion enhances permeation of poorly soluble ABT-102: true supersaturation vs. apparent solubility enhancement.

Amorphous solid dispersions (ASDs) represent a promising formulation approach for poorly soluble drugs. We explored the formulation-related impact of ASDs on permeation rate, apparent solubility and molecular solubility of the poorly soluble drug ABT-102. The influence of fasted state simulated intestinal fluid (FaSSIF) as dispersion medium was also studied. ASDs were prepared by hot-melt extrusion. Permeation rate was assessed by the Caco-2 transwell assay. Cell viability and barrier integrity were assured by AlamarBlue©, TEER and permeability of the hydrophilic marker carboxyfluorescein. Apparent solubility and molecular solubility were evaluated by using centrifugation and inverse dialysis, respectively. The in vitro permeation rate of ABT-102 from aqueous dispersions of the ASD was found 4 times faster than that from the dispersions of the crystals, while apparent solubility and molecular solubility of ABT-102 were increased. Yet, a further increase in apparent solubility due to micellar solubilization as observed when dispersing the ASD in FaSSIF, did not affect molecular solubility or permeation rate. Overall, a good correlation between permeation rate and molecular solubility but not apparent solubility was seen.

Impact of FaSSIF on the solubility and dissolution-/permeation rate of a poorly water-soluble compound.

The poorly water-soluble drug ABT-102, a potent TRPV1 (transient receptor potential cation channel subfamily V member 1) antagonist, was investigated in terms of its solubility and dissolution-permeation rate across Caco-2 cell monolayers in the presence and absence of fasted state simulated intestinal fluid (FaSSIF). ABT-102 showed a more than 30-fold higher apparent solubility in FaSSIF, compared to Hank's balanced salt solution (HBSS). On the other hand, the amount of truly dissolved API in the suspension, as assessed by inverse dialysis, was found hardly influenced by FaSSIF. Neither the drug nor FaSSIF adversely affected cell viability or integrity of the Caco-2 monolayer. P-gp-inhibition experiments confirmed that the drug was not a substrate of the export pump. The flux of ABT-102 across the Caco-2 barrier was found virtually the same in FaSSIF and in buffer, i.e. in vitro overall dissolution-/permeation rate of ABT-102 from suspensions appears not affected by its enhanced apparent solubility due to association with TC/PC-micelles.

In-vitro permeability screening of melt extrudate formulations containing poorly water-soluble drug compounds using the phospholipid vesicle-based barrier.

OBJECTIVES: The phospholipid vesicle-based barrier has recently been introduced as an in-vitro permeation model mimicking gastro-epithelial barriers in terms of passive diffusion of drugs. The aim of this study was to investigate whether the phospholipid vesicle-based barrier was suitable for permeability screening of complex formulations such as solid dispersions. METHODS: Solid dispersions containing the poorly water-soluble drugs HIV-PI 1 (log P=6.2, molar mass=628.80g/mol) and HIV-PI 2 (log P=5.3, molar mass=720.95g/mol), a hydrophilic polymer and different surfactants were tested with respect to their influence on integrity of the barrier in terms of electrical resistance and permeability for calcein. Furthermore, utilisation of a more biologically relevant medium, Hank's balanced salt solution supplemented with Mg(2+) - and Ca(2+) -ions (HBSS (Mg(2+) , Ca(2+) )), has been tested. KEY FINDINGS: Except for the polyoxyl 40 hydrogenated castor oil-containing solid dispersion, no influence on the phospholipid vesicle-based barrier could be observed from the tested samples. Presence of active pharmaceutical ingredients (APIs) in the solid dispersions led to the same results as the corresponding placebo results. First experiments analysing the passive diffusion of both APIs in HBSS (Mg(2+) , Ca(2+) ), evaluated as suitable transport medium, have shown promising results regarding the suitability of the phospholipid vesicle-based barrier for investigation of solid dispersions. CONCLUSIONS: The study indicated that the phospholipid vesicle-based barrier was compatible with selected melt extrudate formulations. The model seemed capable to reveal different transport routes in comparison with Caco-2 cell permeability tests.

In situ formation of nanoparticles upon dispersion of melt extrudate formulations in aqueous medium assessed by asymmetrical flow field-flow fractionation.

In recent years melt extrudates (e.g. Meltrex) have proven to be a promising formulation tool for poorly water-soluble and poorly bioavailable drugs. During the hot-melt extrusion process solid dispersions are formed. For several of these formulations improved bioavailabilities have been reported; the mechanism behind, however is still not very well understood. The aim of this study was to investigate whether solid dispersions prepared by melt extrusion upon dispersion in aqueous medium form particles and/or supramolecular assemblies. The formulation investigated here contained the human immunodeficiency virus (HIV) protease inhibitors lopinavir and ritonavir, polyvinylpyrrolidone-vinyl acetate copolymer (Kollidon VA64), sorbitan monolaurate (Span((R)) 20) and hydrophilic fumed silica (Aerosil 200). The aqueous dispersions originating from both, API-containing and placebo formulation were investigated using photon correlation spectroscopy (PCS) and asymmetrical flow field-flow fractionation (AsFlFFF) with subsequent online multi-angle light-scattering (MALS) particle size analysis. The content of both APIs in the AsFlFFF-fractions was quantified using high performance liquid chromatography-mass spectrometry. PCS indicated sub-micron particles. AsFlFFF revealed the co-existence of up to three different types of colloidal to nanoparticulate assemblies in the aqueous dispersions. Even though a complete resolution of the composition of the sub-fractions could not be achieved, the following types could be clearly distinguished: The first fraction eluting from AsFlFFF, appears to be colloidal polymer. Only marginal amounts of the APIs were found associated with the polymer. Secondly, API-rich nanoparticles eluted. Thirdly, nanoparticulate assemblies assigned to sorbitan monolaurate and/or hydrophilic fumed silica were identified. A limited amount of drug was found associated with this fraction. Using AsFlFFF-MALS the size of particles in fractions could be determined. From this experience AsFlFFF is regarded as promising technique for investigation of particles/structures originating during dispersion of melt extrudates in aqueous medium in terms of size and type of nanoparticles and their API-content.

Novel RGD lipopeptides for the targeting of liposomes to integrin-expressing endothelial and melanoma cells.

RGD peptides targeting alphav-integrins are promising ligands for the generation of vascular targeting agents. We isolated from phage display RGD motif libraries novel high-affinity cyclic RGD peptides by selection on either endothelial or melanoma cells. Although the starting sequences contained only two cysteine residues flanking the RGD motif, several of the isolated peptides possessed four cysteine residues. A high-affinity peptide (RGD10) constrained by only one disulfide bond was used to generate novel lipopeptides composed of a lipid anchor, a short flexible spacer and the peptide ligand conjugated to the spacer end. Incorporation of RGD10 lipopeptides into liposomes resulted in specific and efficient binding of the liposomes to integrin-expressing cells. In vivo experiments applying doxorubicin-loaded RGD10 liposomes in a C26 colon carcinoma mouse model demonstrated improved efficacy compared with free doxorubicin and untargeted liposomes.

Targeting of immunoliposomes to endothelial cells using a single-chain Fv fragment directed against human endoglin (CD105).

We generated immunoliposomes targeting proliferating endothelial cells by chemically coupling a single-chain Fv fragment (scFv A5) directed against human endoglin to the liposomal surface. For this purpose, we introduced an additional cysteine residue at the C-terminus of the scFv fragment. This scFv' fragment was expressed in soluble form in bacteria and allowed for a site-directed coupling to sulfhydryl-reactive lipids incorporated into the lipid bilayer. The immunoliposomes (ILA5) showed rapid and strong binding to human endoglin-expressing endothelial cells (HUVEC, HDMEC), while no binding was observed with various endoglin-negative cell lines and blood lymphocytes. In vitro, ILA5 were stable for several hours in serum- or plasma-containing medium. Incubation of endothelial cells with ILA5 at 37 degrees C led to increased binding and internalisation of the liposomes as evidenced by a perinuclear accumulation. In vitro, doxorubicin-loaded ILA5 showed an increased cytotoxicity towards endothelial cells compared to untargeted liposomes and free doxorubicin. Since the vasculature of tumours is easily accessible to drug carrier systems, the described endothelial cell-specific immunoliposomes may be useful for the development of efficacious and safe vascular targeting agents in cancer therapy.

Isolation from phage display libraries of lysine-deficient human epidermal growth factor variants for directional conjugation as targeting ligands.

Ligand-targeted anticancer therapeutics represent an opportunity for the selective and efficient delivery of drugs to tumours. The chemical coupling of ligands to drugs or drug carrier systems is, however, often hampered by the presence of multiple reactive groups within the ligand, for example, epsilon-NH(2) groups in lysine side chains. In this paper, we describe the isolation by phage display of human epidermal growth factor (EGF) variants without lysine and a reduced number of arginine residues. The selection on A431 carcinoma cells also revealed that R41 is indispensable for EGF binding activity as all EGF variants contained an arginine residue at this position. One EGF variant (EGFm1) with K28Q, R45S, K48S and R53S mutations was expressed in bacteria and showed an identical binding activity as wild-type EGF. EGFm1 could be labelled with fluorescein isothiocyanate demonstrating the accessibility of the N-terminal amino group for coupling reagents. Furthermore, coupling of EGFm1 to PEGylated liposomes resulted in target cell-specific binding and internalization of the liposomes. These human EGF variants should be advantageous for the generation of anticancer therapeutics targeting the EGF receptor, which is overexpressed by a wide variety of different tumours.