Tian- Xiang

Enhanced active liposomal loading of a poorly soluble ionizable drug using supersaturated drug solutions.

Nanoparticulate drug carriers such as liposomal drug delivery systems are of considerable interest in cancer therapy because of their ability to passively accumulate in solid tumors. For liposomes to have practical utility for antitumor therapy in patients, however, optimization of drug loading, retention, and release kinetics are necessary. Active loading is the preferred method for optimizing loading of ionizable drugs in liposomes as measured by drug-to-lipid ratios, but the extremely low aqueous solubilities of many anticancer drug candidates may limit the external driving force, thus slowing liposomal uptake during active loading. This report demonstrates the advantages of maintaining drug supersaturation during active loading. A novel method was developed for creating and maintaining supersaturation of a poorly soluble camptothecin analogue, AR-67 (7-t-butyldimethylsilyl-10-hydroxycamptothecin), using a low concentration of a cyclodextrin (sulfobutylether-β-cyclodextrin) to inhibit crystallization over a 48 h period. Active loading into liposomes containing high concentrations of entrapped sodium or calcium acetate was monitored using drug solutions at varying degrees of supersaturation. Liposomal uptake rates increased linearly with the degree of supersaturation of drug in the external loading solution. A mathematical model was developed to predict the rate and extent of drug loading versus time, taking into account the chemical equilibria inside and outside of the vesicles and the transport kinetics of various permeable species across the lipid bilayer and the dialysis membrane. Intraliposomal sink conditions were maintained by the high internal pH caused by the efflux of acetic acid and exchange with AR-67, which undergoes lactone ring-opening, ionization, and membrane binding in the interior of the vesicles. The highest drug to lipid ratio achieved was 0.17 from a supersaturated solution at a total drug concentration of 0.6 mg/ml. The rate and extent of loading was similar when a different intraliposomal metal cation (sodium) was used instead of calcium. The proposed method may have general application in overcoming the formulation challenges associated with the liposomal delivery of poorly soluble, ionizable anticancer agents.

Stable supersaturated aqueous solutions of silatecan 7-t-butyldimethylsilyl-10-hydroxycamptothecin via chemical conversion in the presence of a chemically modified β-cyclodextrin

AbstractPurpose. A method for obtaining clear supersaturated aqueous solutions for parenteral administration of the poorly soluble experimental anti-cancer drug silatecan 7-t-butyldimethylsilyl-10-hydroxycamptothecin (DB-67) has been developed. Methods. Equilibrium solubilities of DB-67 were determined in various solvents and pH values, and in the presence of chemically modified water-soluble β-cyclodextrins. The stoichiometry and binding constants for complexes of the lactone form of DB-67 and its ring-opened carboxylate with sulfobutyl ether and 2-hydroxypropyl substituted β-cyclodextrins (SBE-CD and HP-CD) were obtained by solubility and circular dichroism spectroscopy, respectively. Kinetics for the reversible ring-opening of DB-67 in aqueous solution and for lactone precipitation were determined by HPLC with UV detection. Results. Solubilities of DB-67 lactone in various injectable solvent systems were found to be at least one order of magnitude below the target concentration (2 mg/ml). DB-67 forms inclusion complexes with SBE-CD and HP-CD but the solubilization attainable is substantially less than the target concentration. Slow addition of DB-67/DMSO into 22.2% (w/v) SBE-CD failed to yield stable supersaturated solutions due to precipitation. Stable supersatured solutions were obtained, however, by mixing a concentrated alkaline aqueous solution of DB-67 carboxylate with an acidified 22.2% (w/v) SBE-CD solution. Ring-closure yielded supersaturated solutions that could be lyophilized and reconstituted to clear, stable, supersaturated solutions. Conclusions. The method developed provides an alternative to colloidal dispersions (e.g., liposomal suspensions, emulsions, etc.) for parenteral administration of lipophilic camptothecin analogs.

Molecular Dynamics Simulation of Amorphous Indomethacin-Poly(Vinylpyrrolidone) Glasses: Solubility and Hydrogen Bonding Interactions

Amorphous drug dispersions are frequently employed to enhance solubility and dissolution of poorly water-soluble drugs and thereby increase their oral bioavailability. Because these systems are metastable, phase separation of the amorphous components and subsequent drug crystallization may occur during storage. Computational methods to determine the likelihood of these events would be very valuable, if their reliability could be validated. This study investigates amorphous systems of indomethacin (IMC) in poly(vinylpyrrolidone) (PVP) and their molecular interactions by means of molecular dynamics (MD) simulations. IMC and PVP molecules were constructed using X-ray diffraction data, and force-field parameters were assigned by analogy with similar groups in Amber-ff03. Five assemblies varying in PVP and IMC composition were equilibrated in their molten states then cooled at a rate of 0.03 K/ps to generate amorphous glasses. Prolonged aging dynamic runs (100 ns) at 298 K and 1 bar were then carried out, from which solubility parameters, the Flory-Huggins interaction parameter, and associated hydrogen bonding properties were obtained. Calculated glass transition temperature (T(g)) values were higher than experimental results because of the faster cooling rates in MD simulations. Molecular mobility as characterized by atomic fluctuations was substantially reduced below the T(g) with IMC-PVP systems exhibiting lower mobilities than that found in amorphous IMC, consistent with the antiplasticizing effect of PVP. The number of IMC-IMC hydrogen bonds (HBs) formed per IMC molecule was substantially lower in IMC-PVP mixtures, particularly the fractions of IMC molecules involved in two or three HBs with other IMC molecules that may be potential precursors for crystal growth. The loss of HBs between IMC molecules in the presence of PVP was largely compensated for by the formation of IMC-PVP HBs. The difference (6.5 MPa(1/2)) between the solubility parameters in amorphous IMC (25.5 MPa(1/2)) and PVP (19.0 MPa(1/2)) suggests a small, positive free energy of mixing, although it is close to the criterion for miscibility (<7 MPa(1/2)). In contrast to the solubility-parameter method, the calculated Flory-Huggins interaction parameter (-0.61 ± 0.25), which takes into account the IMC-PVP interaction energy, predicts complete miscibility at all PVP compositions, in agreement with experimental observations. These results from MD simulations were combined with experimental values for the crystalline γ-polymorph of IMC and amorphous IMC to estimate the solubility of IMC in amorphous PVP dispersions and the theoretical enhancement in the aqueous solubility of IMC molecularly dispersed in PVP at various volume fractions.

Molecular dynamics simulation of amorphous indomethacin.

Molecular dynamics (MD) simulations have been conducted using an assembly consisting of 105 indomethacin (IMC) molecules and 12 water molecules to investigate the underlying dynamic (e.g., rotational and translational diffusivities and conformation relaxation rates) and structural properties (e.g., conformation, hydrogen-bonding distributions, and interactions of water with IMC) of amorphous IMC. These properties may be important in predicting physical stability of this metastable material. The IMC model was constructed using X-ray diffraction data with the force-field parameters mostly assigned by analogy with similar groups in Amber-ff03 and atomic charges calculated with the B3LYP/ccpVTZ30, IEFPCM, and RESP models. The assemblies were initially equilibrated in their molten state and cooled through the glass transition temperature to form amorphous solids. Constant temperature dynamic runs were then carried out above and below the T(g) (i.e., at 600 K (10 ns), 400 K (350 ns), and 298 K (240 ns)). The density (1.312 ± 0.003 g/cm(3)) of the simulated amorphous solid at 298 K was close to the experimental value (1.32 g/cm(3)) while the estimated T(g) (384 K) was ~64 degrees higher than the experimental value (320 K) due to the faster cooling rate. Due to the hindered rotation of its amide bond, IMC can exist in different diastereomeric states. Different IMC conformations were sufficiently sampled in the IMC melt or vapor, but transitions occurred rarely in the glass. The hydrogen-bonding patterns in amorphous IMC are more complex in the amorphous state than in the crystalline polymorphs. Carboxylic dimers that are dominant in α- and γ-crystals were found to occur at a much lower probability in the simulated IMC glasses while hydrogen-bonded IMC chains were more easily identified patterns in the simulated amorphous solids. To determine molecular diffusivity, a novel analytical method is proposed to deal with the non-Einsteinian behavior, in which the temporal evolution of the apparent diffusivity D(t) is described by a relaxation model such as the KWW function and extrapolated to infinite time. The diffusion coefficient found for water diffusing in amorphous indomethacin at 298 K (2.7 × 10(-9) cm(2)/s) compares favorably to results obtained in experimental IMC glasses (0.9-2.0 × 10(-9) cm(2)/s) and is mechanistically associated with β-relaxation processes that are dominant in sub-T(g) glasses.

A Computer Simulation of Functional Group Contributions to Free Energy in Water and a DPPC Lipid Bilayer

A series of all-atom molecular dynamics simulations has been performed to evaluate the contributions of various functional groups to the free energy of solvation in water and a dipalmitoylphospatidylcholine lipid bilayer membrane and to the free energies of solute transfer (Delta(DeltaG(o))X) from water into the ordered-chain interior of the bilayer. Free energies for mutations of the alpha-H atom in p-toluic acid to six different substituents (-CH3, -Cl, -OCH3, -CN, -OH, -COOH) were calculated by a combined thermodynamic integration and perturbation method and compared to literature results from vapor pressure measurements, partition coefficients, and membrane transport experiments. Convergence of the calculated free energies was indicated by substantial declines in standard deviations for the calculated free energies with increased simulation length, by the independence of the ensemble-averaged Boltzmann factors to simulation length, and the weak dependence of hysteresis effects on simulation length over two different simulation lengths and starting from different initial configurations. Calculated values of Delta(DeltaG(o))X correlate linearly with corresponding values obtained from lipid bilayer transport experiments with a slope of 1.1 and from measurements of partition coefficients between water and hexadecane or decadiene, with slopes of 1.1 and 0.9, respectively. Van der Waals interactions between the functional group of interest and the acyl chains in the ordered chain region account for more than 95% of the overall potential energy of interaction. These results support the view that the ordered chain region within the bilayer interior is the barrier domain for transport and that solvation interactions within this region resemble those occurring in a nonpolar hydrocarbon.

Mechanistic model and analysis of doxorubicin release from liposomal formulations.

Reliable and predictive models of drug release kinetics in vitro and in vivo are still lacking for liposomal formulations. Developing robust, predictive release models requires systematic, quantitative characterization of these complex drug delivery systems with respect to the physicochemical properties governing the driving force for release. These models must also incorporate changes in release due to the dissolution media and methods employed to monitor release. This paper demonstrates the successful development and application of a mathematical mechanistic model capable of predicting doxorubicin (DXR) release kinetics from liposomal formulations resembling the FDA-approved nanoformulation DOXIL® using dynamic dialysis. The model accounts for DXR equilibria (e.g. self-association, precipitation, ionization), the change in intravesicular pH due to ammonia release, and dialysis membrane transport of DXR. The model was tested using a Box-Behnken experimental design in which release conditions including extravesicular pH, ammonia concentration in the release medium, and the dilution of the formulation (i.e. suspension concentration) were varied. Mechanistic model predictions agreed with observed DXR release up to 19h. The predictions were similar to a computer fit of the release data using an empirical model often employed for analyzing data generated from this type of experimental design. Unlike the empirical model, the mechanistic model was also able to provide reasonable predictions of release outside the tested design space. These results illustrate the usefulness of mechanistic modeling to predict drug release from liposomal formulations in vitro and its potential for future development of in vitro - in vivo correlations for complex nanoformulations.

Water Uptake, Distribution, and Mobility in Amorphous Poly(d,l‐Lactide) by Molecular Dynamics Simulation

An explicit all-atom computational model for amorphous poly(lactide) (PLA) was developed. Molecular dynamics simulations of PLA glasses were conducted to explore various molecular interactions and predict certain physical properties. The density of a newly formed PLA glass aged for 100 ns at 298 K was 1.23 g/cm(3), close to the experimental range (1.24-1.25 g/cm(3)). The glass transition temperature (Tg = 364 K) was higher than experimental values because of the fast cooling rate (0.03 K/ps) in the simulation. The solubility parameter (20.6 MPa(1/2)) compared favorably to the literature. The water sorption isotherm obtained by relating the excess chemical potential of water in PLA to the Henrys law constant for water sorption was close to the experiment. At 0.6% (w/w), water molecules localize next to polar ester groups in PLA because of hydrogen bonding. Local mobility in PLA as characterized by the atomic fluctuation was sharply reduced near the Tg , decreasing further with aging at 298 K. The non-Einsteinian diffusion of water was found to correlate with the rotational β-relaxation of PLA C=O groups at 298 K. A relaxation-diffusion coupling model proposed recently by the authors gave a diffusion coefficient (1.3 × 10(-8) cm(2) /s at 298 K) which is comparable to reported experimental values.

Development of structure-lipid bilayer permeability relationships for peptide-like small organic molecules.

Computational methods to estimate passive membrane permeability coefficients of organic molecules, including peptides, would be valuable in understanding various biological processes associated with molecular transport across cell membranes and in reducing the time required for screening developability properties of new drug candidates. This study explores the suitability of fragment-based linear free energy relationships (LFERs) to predict lipid bilayer permeability coefficients and decadiene/water partition coefficients of a set of 47 model permeants. The inclusion of mono-, di-, and tripeptides comprised of glycine, alanine, and sarcosine residues in the database presented added challenges due to the apparent lack of independence of the contribution of the backbone amide residue in peptides to the free energy of transfer (Delta(Delta G degrees ) -CONH-) from water to organic solvents or to the bilayer barrier domain. In order to elucidate the impact of neighboring group effects on Delta(Delta G degrees ) -CONH-, a series of RGZ glycine (G)-containing peptides having an additional -NHCH 2CO- residue compared to their RZ counterparts were synthesized, where R = acetyl (Ac-), 4-carboxymethylphenyl acetyl (CMPA-), or 4-methylphenyl acetyl (MPA-), and Z = -OH, -OMe, -NHMe, or -NMe 2. While variations in R had no significant impact on Delta(Delta G degrees ) -Gly-, significant effects of neighboring ( i + 1) Z substituents at the C-terminus were revealed both in studies of the relative transport of RGZ/RZ compound pairs across DOPC bilayers and partitioning between water and 1,9-decadiene (a bulk solvent with a similar chemical selectivity to the barrier domain of DOPC/eggPC bilayers). The proximity effects decline when the bulk solvent used in partitioning studies is 1-octanol, suggesting a possible role for intramolecular hydrogen bonding in the observed nonadditivity of Delta(Delta G degrees ) -CONH-. A new LFER for predicting decadiene/water partition coefficients was developed by including the contributions of polar fragments, total nonpolar surface area of nonpolar fragments, and correction factors to account for the effects of i + 1 substituents in peptides on the group contribution of the peptide backbone amide bond to the free energy of transfer. This LFER could be used to predict lipid bilayer permeability coefficients by including an additional term to account for the added influence of molecular size on bilayer permeability.

Determination of Key Parameters for a Mechanism-Based Model to Predict Doxorubicin Release from Actively Loaded Liposomes

Despite extensive study of liposomal drug formulations, reliable predictive models of release kinetics in vitro and in vivo are still lacking. Progress in the development of robust, predictive release models has been hindered by a lack of systematic, quantitative characterization of these complex drug delivery systems with respect to the myriad of factors that may influence drug release kinetics and the wide range of dissolution media/methods employed to monitor release. In this paper, the key processes and parameters needed to develop a complete mechanism-based model for doxorubicin release from actively loaded liposomal formulations resembling Doxil(®) are determined. Quantitative models must account for the driving force(s) [i.e., activity gradient(s) of the permeable species between the intraliposomal and external media] and the permeability-area product(s) for lipid bilayer transport. These factors are intertwined as membrane permeability-area products require knowledge of the drug species and concentrations that account for the release. The necessary information includes values for the drug pKa, identity of the permeable species and species permeability coefficients, a model to describe drug self-association and the relevant equilibrium constant(s), the bilayer/water partition coefficient of the predominant drug species under relevant pH conditions, and the solubility product (Ksp ) for intraliposomal precipitates that exist in such formulations.

Factors Affecting the In Vivo Lactone Stability and Systemic Clearance of the Lipophilic Camptothecin Analogue AR-67

ABSTRACTPurposeThe narrow efficacy-toxicity window of anticancer agents necessitates understanding of factors contributing to their disposition. This is especially true for camptothecins as they exist in the lactone and carboxylate forms with each moiety differentially interacting with efflux or uptake transporters. Here we determined the disposition of the lactone and carboxylate forms of AR-67, a 3rd generation camptothecin analogue.MethodsPharmacokinetic studies were conducted in rats given intravenous boluses of AR-67 lactone or carboxylate with or without pharmacologic inhibitor pretreatment (GF120918 or rifampin). Pharmacokinetic modeling was used to estimate clearances, while simulations assessed the impact of clearance changes on overall AR-67 exposure.ResultsOur modeling showed that carboxylate clearance was 3.5-fold higher than that of the lactone. GF120918 decreased lactone clearance only, but rifampin decreased both lactone and carboxylate clearances. Simulations showed that decreasing carboxylate clearance, which controls the overall drug disposition, leads to significant increase in AR-67 exposure.ConclusionThe apparent in vivo blood stability of AR-67 is partly dependent on the increased carboxylate clearance. This may have clinical implications for populations with single nucleotide polymorphisms that impair the function of uptake transporter genes (e.g., SLCO1B1), which are potentially responsible for AR-67 carboxylate clearance.