The effects of spray drying, HPMCAS grade, and compression speed on the compaction properties of itraconazole-HPMCAS spray dried dispersions.

Spray dried dispersions (SDDs) have the potential to dramatically improve the oral bioavailability of drugs with poor water solubility. However, SDDs tend to have material attributes, such as small particle size, low bulk density, and poor flowability, which are undesirable for downstream processing such as tableting. The objective was to perform a comprehensive compaction characterization of both physical mixtures and SDDs consisting of itraconazole (ITZ) and hypromellose acetate succinate (HPMCAS) to elucidate process and material influences on compressibility and compactibility. We fabricated SDDs with 20% ITZ as a model BCS Class 2 drug and 80% HPMCAS as a polymer carrier. Results indicate that SDDs, as well physical mixtures of ITZ and HPMCAS, were easily deformable with similar compressibility profiles across all compression speeds. Analysis of Heckel plots revealed that yield pressures were fairly low for both physical mixtures and SDDs (43.97-59.75 MPa), indicative of ductile materials. SDDs had a much greater propensity to laminate, especially at higher compression speeds, compared to physical mixtures. This difference is likely due to the higher elastic recovery of SDDs. However, for intact tablets, the mechanical strength of compacts from SDDs tended to be higher than those produced from physical mixtures, likely due to the much smaller particle size of the SDDs. Importantly, examination of the compacts with differential scanning calorimetry did not detect any drug crystallization as a result of compaction. In conclusion, while spray drying did not significantly alter the compressibility of binary mixtures ITZ and HPMCAS, it dramatically impacted compactibility and tabletability, increasing elastic recovery, and making the mixtures more prone to lamination. However, at low compression speeds, SDDs produced tablets with higher tensile strength than physical mixtures.

Fragment-based screening using surface plasmon resonance technology.

Surface plasmon resonance (SPR) technology has emerged as a new and powerful technique to investigate the interaction between low-molecular-weight molecules and target proteins. In the present work, the authors assemble from a large compound collection a library of 2226 molecules (fragments having low molecular weights between 100 and 300 Da) to screen them for binding to chymase, a serine protease. Both the active chymase and a zymogen-like form of the protein were used in parallel to distinguish between specific and unspecific binding. The relative ligand-binding activity of the immobilized protein was periodically measured with a reference compound. The screening experiments were performed at 25 degrees C at a fragment concentration of 200 microM in the presence of 2% DMSO. Applying the filter cascade, affinity-selectivity-competition (competition with reference compounds and cross-competition with fragments), 80 compounds show up as positive screening hits. Competition experiments between fragments show that they bind to different parts of the active site. Of 36 fragments co-crystallized for X-ray studies, 12 could be located in the active site of the protein. These results validate the authors' library and demonstrate that the application of SPR technology as a filter in fragment screening can be achieved successfully.

Evaluation of the deformation behavior of binary systems of methacrylic acid copolymers and hydroxypropyl methylcellulose using a compaction simulator.

Methacrylic acid copolymers have been shown to enhance release of weakly basic drugs from rate controlling polymer matrices through the mechanism of microenvironmental pH modulation. Since these matrices are typically formed through a compaction process, an understanding of the deformation behavior of these polymers in there neat form and in combination with rate controlling polymers such as HPMC is critical to their successful formulation. Binary mixes of two methacrylic acid copolymers, Eudragit L100 and L100-55 in combination with HPMC K4M were subjected to compaction studies on a compaction simulator. The deformation behavior of the powder mixes was analyzed based on pressure-porosity relationships, strain rate sensitivity (SRS), residual die wall force data and work of compaction. Methacrylic acid copolymers, L100-55 and L-100 and the hydrophilic polymer, HPMC K4M exhibited Heckel plots representative of plastic deformation although L-100 exhibited significantly greater resistance to densification as evident from the high yield pressure values ( approximately 120MPa). The yield pressures for the binary mixes were linearly related to the weight fractions of the components. All powder mixes exhibited significant speed sensitivity with SRS values ranging from 21.7% to 42.4%. The residual die-wall pressures indicated that at slow speeds (1mm/s) and at lower pressures (<150MPa), HPMC possesses significant elastic behavior. However, the good compacts formed at this punch speed indicate significant plastic deformation and bond formation which is able to predominate over the elastic recovery component. The apparent mean yield pressure values, the residual die-wall forces and the net work of compaction exhibited a linear relationship with mixture composition, thereby indicating predictability of these parameters based on the behavior of the neat materials.

Predicting plasma protein binding of drugs--revisited.

Plasma protein binding of drugs has been studied for almost 100 years, but despite the accumulation of large amounts of data, the accurate prediction of this ADME parameter continues to be problematic. This review outlines recent efforts on the development of prediction tools for plasma protein binding of drugs, specifically human serum albumin, in the context of its relevance and its influencing factors. The issue of why it is difficult to achieve prediction of sufficient quality for a diverse dataset will also be considered.

Predicting plasma protein binding of drugs: a new approach.

In spite of the large amount of plasma protein binding data for drugs, it is not obvious and there is no clear consensus among different disciplines how to deal with this parameter in multidimensional lead optimization strategies. In this work, we have made a comprehensive study on the importance of plasma protein binding and the influencing factors in order to get new insights for this molecular property. Our analysis of the distribution of percentage plasma protein binding among therapeutic drugs showed that no general rules for protein binding can be derived, except for the class of chemotherapeutics, where a clear trend towards lower binding could be observed. For the majority of indication areas, however, empirical rules are missing. We present here an extensive list of multiply determined primary association constants for binding to human serum albumin (HSA) for 138 compounds from the literature. Correlating these binding constants with the percentage fraction of protein bound showed that the percentage data above 90%, corresponding to a binding constant below 6 microM, are of insufficient accuracy. Furthermore, it could be demonstrated that the lipophilicity of drugs, traditionally felt to dominate binding to HSA, is not the only relevant descriptor. Here, we report a generic model for the prediction of drug association constants to HSA, which uses a pharmacophoric similarity concept and partial least square analysis (PLS) to construct a quantitative structure-activity relationship. It is able to single out the submicromolar to nanomolar binders, i.e. to differentiate between 99.0 and 99.99% plasma protein binding. Depending on the system, this can be important in medicinal chemistry programs and may together with other computed physicochemical and ADME properties assist in the prioritization of synthetic strategies.

Solid-state properties of warfarin sodium 2-propanol solvate.

The goal of the present work was to understand the effect of relative humidity (RH) and temperature on the molecular structure, crystal structure, and physical properties of warfarin sodium 2-propanol solvate (W). After previous determination of the crystal structure of W, which corresponds to a 1:1 2-propanol solvate, the present work shows that W has a critical RH (60% < RH(0) < or = 68%), below which minimal uptake of water occurs, due to surface adsorption, but above which gradual and continuous uptake of water occurs, due to deliquescence. Deliquescence begins at the surface and proceeds inward into the bulk of the crystal. Single crystal X-ray diffractometry indicates no change in the crystal and molecular structure of W during the initial stages of deliquescence. Studies of the unit cell and volume computations of W show that water can neither find space to enter the crystal lattice, nor can replace 2-propanol. Thus, water does not exchange with 2-propanol within the lattice, contrary to previous reports. Storage of single crystals of W at 120 degrees C for 23 h produces shrinkage cracks along the needle (b) axis, which are interpreted as a reduction in d-spacing of the 00l planes. Thus, under thermal stress, W crystals undergo amorphization with concurrent loss of 2-propanol, which may proceed via an intermediate crystalline phase. The phase changes of W, which depend on RH and temperature, are explained at the molecular level.

Dehydration kinetics of piroxicam monohydrate and relationship to lattice energy and structure.

The dehydration kinetics of piroxicam monohydrate (PM) is analyzed by both model-free and model-fitting approaches. The conventional model-fitting approach assuming a fixed mechanism throughout the reaction is found to be too simplistic. The model-free approach allows for a change of mechanism and activation energy, Ea, during the course of a reaction and is therefore more realistic. The complexity of the dehydration of PM is illustrated by the dependence of Ea on both the heating conditions, isothermal or nonisothermal, and on the fraction of conversion, alpha (0 < or = alpha < or = 1). Under both isothermal and nonisothermal conditions, Ea increases with alpha for 0 < or = alpha < or = 0.25, followed by an approximately constant value of Ea during further dehydration. In the constant-Ea region, isothermal dehydration follows the two-dimensional phase boundary model (R2), whereas nonisothermal dehydration follows a mechanism intermediate between two- and three-dimensional diffusion that cannot be described by any of the common models. Structural studies suggest that the complex hydrogen-bond pattern in PM is responsible for the observed dehydration behavior. Ab initio calculations provide an explanation for the changes in the molecular and crystal structures accompanying the reversible change in hydration state between anhydrous piroxicam Form I and PM. This work also demonstrates the utility of model-free analysis in describing complex dehydration kinetics.

Evaluation of the plug formation process of silicified microcrystalline cellulose.

To investigate the powder plug formation process of silicified microcrystalline cellulose (SMCC) under compression forces consistent with automatic capsule-filling machines, a single-ended saw-tooth wave was used to make powder plugs with different heights (6, 8, 12 mm), at two different punch speeds (1 and 50 mm/s) on a tablet compaction simulator. SMCC was compared to Starch 1500, anhydrous lactose (direct tableting grade), and microcrystalline cellulose. Heckel analysis showed that 'apparent mean yield pressures' (AMYP) of all tested materials increased with an increase in the plug height and punch speed. AMYP appeared to depend on the material type and punch speed. Not all materials fit the Shaxby-Evans relationship at such low compression forces (less than 250 N). Only SMCC 90, SMCC HD90 and anhydrous lactose data fit the equation at both punch speeds. Due to poor axial load transmission, the R values of all tested materials decreased with an increase in the plug height. The experimental data fit the Kawakita equation quite well. Overall, Kawakita's b values were inversely related to AMYP values. The maximum breaking force (MBF) of a 12 mm plug formed at a punch speed of 50 mm/s correlated well with the work of compaction, except for SMCC HD90 and SMCC X, which exhibited very high MBF values. This research demonstrated that several grades of SMCC produced plugs having higher MBF than anhydrous lactose and Starch 1500 under similar compression conditions. The apparently higher compactability of these materials at low plug formation forces may be beneficial in developing direct fill formulations for automatic capsule filling machines.

Summary workshop report: Facilitating oral product development and reducing regulatory burden through novel approaches to assess bioavailability/bioequivalence.

This summary workshop report highlights presentations and over-arching themes from an October 2011 workshop. Discussions focused on best practices in the application of biopharmaceutics in oral drug product development and evolving bioequivalence approaches. Best practices leverage biopharmaceutic data and other drug, formulation, and patient/disease data to identify drug development challenges in yielding a successfully performing product. Quality by design and product developability paradigms were discussed. Development tools include early development strategies to identify critical absorption factors and oral absorption modeling. An ongoing theme was the desire to comprehensively and systematically assess risk of product failure via the quality target product profile and root cause and risk analysis. However, a parallel need is reduced timelines and fewer resources. Several presentations discussed applying Biopharmaceutics Classification System (BCS) and in vitro-in vivo correlations in development and in post-development and discussed both resource savings and best scientific practices. The workshop also focused on evolving bioequivalence approaches, with emphasis on highly variable products (HVDP), as well as specialized modified-release products. In USA, two bioequivalence approaches for HVDP are the reference-scaled average bioequivalence approach and the two-stage group-sequential design. An adaptive sequential design approach is also acceptable in Canada. In European Union, two approaches for HVDP are a two-stage design and an approach to widen C (max) acceptance limits. For some specialized modified-release products, FDA now requests partial area under the curve. Rationale and limitations of such metrics were discussed (e.g., zolpidem and methylphenidate). A common theme was the benefit of the scientific and regulatory community developing, validating, and harmonizing newer bioequivalence methodologies (e.g., BCS-based waivers and HVDP trial designs).

Structure-assisted discovery of an aminothiazole derivative as a lead molecule for inhibition of bacterial fatty-acid synthesis.

Fatty-acid synthesis in bacteria is of great interest as a target for the discovery of antibacterial compounds. The addition of a new acetyl moiety to the growing fatty-acid chain, an essential step in this process, is catalyzed by beta-ketoacyl-ACP synthase (KAS). It is inhibited by natural antibiotics such as cerulenin and thiolactomycin; however, these lack the requirements for optimal drug development. Structure-based biophysical screening revealed a novel synthetic small molecule, 2-phenylamino-4-methyl-5-acetylthiazole, that binds to Escherichia coli KAS I with a binding constant of 25 microM as determined by fluorescence titration. A 1.35 A crystal structure of its complex with its target reveals noncovalent interactions with the active-site Cys163 and hydrophobic residues of the fatty-acid binding pocket. The active site is accessible through an open conformation of the Phe392 side chain and no conformational changes are induced at the active site upon ligand binding. This represents a novel binding mode that differs from thiolactomycin or cerulenin interaction. The structural information on the protein-ligand interaction offers strategies for further optimization of this low-molecular-weight compound.

Structural and biophysical characterization of the 40 kDa PEG-interferon-alpha2a and its individual positional isomers.

The human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) to treat various types of cancer and viral diseases including Hepatitis B/C infections. To improve the pharmacological properties of the drug, a new pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side chain of IFNalpha(2a). (40)PEG-IFNalpha(2a) is a mixture of mainly six monopegylated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively. Here we report the detailed structural and biophysical characterization of (40)PEG-IFNalpha(2a) and its positional isomers, in comparison with IFNalpha(2a), using NMR spectroscopy, analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. Our results show that the three-dimensional structure of IFNalpha(2a) is not modified by the presence of the polymer in all positional isomers constituting (40)PEG-IFNalpha(2a). Regardless of where the PEG-polymer is attached, it adopts a very mobile and flexible random coil conformation, producing a shield for the protein without a permanent coverage of the protein surface. Hydrodynamic data indicate that the protein-attached PEG has a slightly more compact random-coil structure than the free PEG-polymer. Our results also provide evidence of significant structural and physicochemical advantages conferred by the pegylation: increase of the effective hydrodynamic volume and modification of the molecular shape, higher temperature stability, and reduced tendency for aggregation. These results are of tremendous pharmacological interest and benefit as was clinically shown with PEGASYS. This study constitutes a new standard for the characterization of pegylated proteins and enables an important step toward the understanding on a molecular level of the binding of (40)PEG-IFNalpha(2a) and its positional isomers to its cellular receptors.

Crystal engineering yields crystals of cyclophilin D diffracting to 1.7 A resolution.

In the pharmaceutical industry, knowledge of the three-dimensional structure of a specific target facilitates the drug-discovery process. Despite possessing favoured analytical properties such as high purity and monodispersion in light scattering, some proteins are not capable of forming crystals suitable for X-ray analysis. Cyclophilin D, an isoform of cyclophilin that is expressed in the mitochondria, was selected as a drug target for the treatment of cardiac disorders. As the wild-type enzyme defied all attempts at crystallization, protein engineering on the enzyme surface was performed. The K133I mutant gave crystals that diffracted to 1.7 A resolution using in-house X-ray facilities and were suitable for soaking experiments. The crystals were very robust and diffraction was maintained after soaking in 25% DMSO solution: excellent conditions for the rapid analysis of complex structures including crystallographic fragment screening.

Mechanochromism of piroxicam accompanied by intermolecular proton transfer probed by spectroscopic methods and solid-phase changes.

Structural and solid-state changes of piroxicam in its crystalline form under mechanical stress were investigated using cryogenic grinding, powder X-ray diffractometry, diffuse-reflectance solid-state ultraviolet-visible spectroscopy, variable-temperature solid-state (13)C nuclear magnetic resonance spectroscopy, and solid-state diffuse-reflectance infrared Fourier transform spectroscopy. Crystalline piroxicam anhydrate exists as colorless single crystals irrespective of the polymorphic form and contains neutral piroxicam molecules. Under mechanical stress, these crystals become yellow amorphous piroxicam, which has a strong propensity to recrystallize to a colorless crystalline phase. The yellow color of amorphous piroxicam is attributed to charged piroxicam molecules. Variable-temperature solid-state (13)C NMR spectroscopy indicates that most of the amorphous piroxicam consists of neutral piroxicam molecules; the charged species comprise only about 8% of the amorphous phase. This ability to quantify the fractions of charged and neutral molecules of piroxicam in the amorphous phase highlights the unique capability of solid-state NMR to quantify mixtures in the absence of standards. Other compounds of piroxicam, which are yellow, are known to contain zwitterionic piroxicam molecules. The present work describes a system in which proton transfer accompanies both solid-state disorder and a change in color induced by mechanical stress, a phenomenon which may be termed mechanochromism of piroxicam.

Development and evaluation of a miniaturized procedure for determining the bonding index: a novel prototype for solid dosage formulation development.

The purpose of this study was a comparative evaluation of miniaturized vs. University of Minnesota's (i.e., U of Minn. = Hiestand's) procedures for determining the tensile strength, indentation hardness, and bonding index (BI), one out of three Indices of Tableting Performance (ITP). Tableting properties of six direct compression placebo formulations were determined by using a compaction simulator and a Texture Analyser TA-XT2I, or by following the U of Minn. method, which included a specially designed triaxial compression device, a computer-controlled mechanical stress-strain analyzer, and a dynamic pendulum impact apparatus. Miniaturization of the procedures to determine the ITP, as well as the ability to differentiate between materials while operating compaction cycles more comparable to standard tablet production conditions, enabled proper evaluation of each material's inherent tableting properties. Indentation diameter calculated via an empirical equation appeared to correlate well with, and provided acceptable precision of accuracy to, the determination of indentation diameter via standard optical microscopy methods. The miniaturized and U of Minn. procedure exhibited a significant degree of correlation when comparing the BI. However, the tensile strength and indentation hardness values were somewhat different due to the use of triaxial decompression for the U of Minn. procedure vs. standard compaction profiling for the miniaturized procedure. The present direct compression placebo formulation data gathered from the miniaturized procedures and compared with the U of Minn. method for determining the ITP suggest that both techniques yield similar conclusions. However, discrimination of out-of-die compaction properties determined via the miniaturized procedures, such as tensile strength and indentation hardness, appeared to associate more precisely with changes in strain rate, thus allowing better discrimination of particle-particle interactions and ductile-to-brittle characteristics as a function of compaction speed and pressure.

SPR-based interaction studies with small molecular weight ligands using hAGT fusion proteins.

An immobilization procedure for protein on surface plasmon resonance sensor (SPR) chips is described. The target protein, cyclophilin D, is thereby genetically linked to a mutant of the human DNA repair protein O(6)-alkylguanine-DNA-alkyltransferase (hAGT). The procedure includes the immobilization of an alkylguanine derivative on the surface by amine coupling and contact of the surface with a solution of the fusion protein (TCypD-hAGT). TCypD-hAGT could be immobilized using buffer solutions of purified protein or cell extracts. High densities of covalently linked proteins were achieved by either procedure. Binding experiments performed with the ligand cyclosporin A indicate relative binding activities close to 100%. The K(D) value (12 nM) and the kinetic rate constants k(on) (3 x 10(5)M(-1)s(-1)) and k(off) (4 x 10(-3)s(-1)) are given and compared to values determined for cyclophilin D linked to the surface by amide coupling chemistry. The K(D) value is in excellent agreement with the K(D) value determined in solution by fluorescence titration.

The monotopic membrane protein human oxidosqualene cyclase is active as monomer.

The monotopic integral membrane protein 2,3-oxidosqualene cyclase (OSC) catalyzes the formation of lanosterol the first sterol precursor of cholesterol in mammals. Therefore, it is an important target for the development of new hypocholesterolemic drugs. Here, we report the overexpression and purification of functional human OSC (hOSC) in Pichia pastoris. The obtained IC(50) for the reference inhibitor Ro 48-8071 is nearly identical for the recombinant hOSC compared to OSC from human liver microsomes. The correlation of analytical ultracentrifugation data and activity measurements showed the highest enzymatic activity for the monomeric hOSC indicating that this would be the natural form. Furthermore, these data helped us to identify the detergent for a successful crystallization of the protein. The availability of this active recombinant human membrane protein is a very important step on the way to a more detailed functional and structural characterization of OSCs.