Search

Atomic force microscopy (AFM) and modulated differential scanning calorimetry (mDSC) were used to evaluate the extent of mixing of a hot melt extrusion process for producing solid dispersions of copovidone and D-α-tocopherol polyethylene glycol 1000 succinate (TPGS 1000). In addition to composition, extrusion process parameters of screw speed and thermal quench rate were varied. The data indicated that for 10% TPGS and 300 rpm screw speed, the mixing was insufficient to yield a single-phase amorphous material. AFM images of the extrudate cross section for air-cooled material indicate round domains 200 to 700 nm in diameter without any observed alignment resulting from the extrusion whereas domains in extrudate subjected to chilled rolls were elliptical in shape with uniform orientation. Thermal analysis indicated that the domains were predominantly semi-crystalline TPGS. For 10% TPGS and 600 rpm screw speed, AFM and mDSC data were consistent with that of a single-phase amorphous material for both thermal quench rates examined. When the TPGS concentration was reduced to 5%, a single-phase amorphous material was achieved for all conditions even the slowest screw speed studied (150 rpm).

Peptides are an important class of endogenous ligands that regulate key biological cascades. As such, peptides represent a promising therapeutic class with the potential to alleviate many severe disease states. Despite their therapeutic potential, peptides frequently pose drug delivery challenges to scientists. This review introduces the physicochemical, biophysical, biopharmaceutical, and formulation developability aspects of peptides pertinent to the drug discovery-to-development interface. It introduces the relevance of these properties with respect to the delivery modalities available for peptide pharmaceuticals, with the parenteral route being the most prevalent route of administration. This review also presents characterization strategies for oral delivery of peptides with the aim of illuminating developability issues with the drug candidate. A brief overview of other routes of administration, including inhaled, transdermal, and intranasal routes, is provided as these routes are generally preferred by patients over injectables. Finally, this review presents formulation techniques to mitigate some of the developability obstacles associated with peptide delivery. The authors emphasize opportunities for the thoughtful application of pharmaceutical science to the development of peptide drugs and to the general advancement of this promising class of pharmaceuticals.

The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.

Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.

Ionizable amino lipids are being pursued as an important class of materials for delivering small interfering RNA (siRNA) therapeutics, and research is being conducted to elucidate the structure-activity relationships (SAR) of these lipids. The pK(a) of cationic lipid headgroups is one of the critical physiochemical properties of interest due to the strong impact of lipid ionization on the assembly and performance of these lipids. This research focused on developing approaches that permit the rapid determination of the relevant pK(a) of the ionizable amino lipids. Two distinct approaches were investigated: (1) potentiometric titration of amino lipids dissolved in neutral surfactant micelles; and (2) pH-dependent partitioning of a fluorescent dye to cationic liposomes formulated from amino lipids. Using the approaches developed here, the pK(a) values of cationic lipids with distinct headgroups were measured and found to be significantly lower than calculated values. It was also found that lipid-lipid interaction has a strong impact on the pK(a) values of lipids. Lysis of model biomembranes by cationic lipids was used to evaluate the impact of lipid pK(a) on the interaction between cationic lipids and cell membranes. It was found that cationic lipid-biomembrane interaction depends strongly on lipid pK(a) and solution pH, and this interaction is much stronger when amino lipids are highly charged. The presence of an optimal pK(a) range of ionizable amino lipids for siRNA delivery was suggested based on these results. The pK(a) methods reported here can be used to support the SAR screen of cationic lipids for siRNA delivery, and the information revealed through studying the impact of pK(a) on the interaction between cationic lipids and cell membranes will contribute significantly to the design of more efficient siRNA delivery vehicles.

The crystallization of Etoricoxib, a polymorphic compound, has been optimized and controlled by seeding with the desired polymorph at a moderate supersaturation condition. To enhance the process robustness, near infrared spectroscopy (NIRS) has been evaluated as an inline measurement method for the concentration of Etoricoxib prior to seeding in the crystallization process. In this NIRS method, a spectral discriminant analysis based on principal component analysis (PCA) was established to detect the presence of solids produced by premature crystallization, or bubbles in the path of light. Once a spectrum was qualified as that of clear solution, concentration of Etoricoxib was calculated by a NIRS calibration model built with partial least squares (PLS) regression and with offline HPLC analysis as the reference method. This model was accurate with a standard error of cross validation (SECV) less than 1.2 mg/g Etoricoxib and a standard error of prediction (SEP) less than 1.7 mg/g over the concentration range from 50 to 170 mg/g, temperature range from 49 to 65 degrees C, and different sources of materials. In addition, all aspects of the offline HPLC method, especially the sampling procedure, were optimized to provide an accurate reference for NIRS calibration models. The application of this method at a pilot plant has demonstrated its capability of accurately measuring the process concentration of Etoricoxib as well as detecting the presence of solids produced by premature crystallization before seeding.

High-intensity ultrasonic treatment of MeOH/H2O, IPA/H2O, EtOH or H2O suspensions of C-14 labeled and unlabeled pharmaceuticals successfully reduced the average particle diameter from a range of 8–260 to 4–15 µm as determined by optical microscopy. Copyright © 2004 John Wiley & Sons, Ltd.

Anhydrate and hemihydrate phases of etoricoxib (5-chloro-6‘-methyl-3-[4-(methylsulfonyl)phenyl]-2,3‘-bipyridine) are described. The ternary phase diagram of etoricoxib, isopropyl acetate, and water...

Four anhydrous polymorphic forms (I, II, III and IV) of an NK1 receptor antagonist, Compound A, have been discovered. The pure compound can exist as either Forms I or II at room temperature and Forms III or IV at elevated temperatures. The four polymorphs were characterized by differential scanning calorimetry (DSC), thermogravimetric analysis, X-ray powder diffraction (XRPD) and solid-state NMR spectroscopy (SSNMR). Polymorphic transformations in the solid phase were studied using DSC, hot stage XRPD, temperature-modulated SSNMR and hot stage optical microscopy. The solubilities of Forms I and II in tert-butyl acetate at different temperatures were measured and the relative stability of the two forms was established. The thermodynamic transformation temperatures between Forms I and III, as well as Forms II and IV, were estimated by DSC. Transformation from Form III to IV, which is undetectable in a normal calorimetric run, was revealed through careful thermal programming. An interesting conversion route from Form I, a more stable form at room temperature, to Form II, a less stable form at room temperature was discovered.

The mechanism of chiral selectivity of a difunctional polymer imprinted with dansyl-L-phenylalanine was investigated using the polymer as an HPLC stationary phase. Temperature studies revealed that the mass transfer of the imprinted enantiomer with the polymer was sluggish at low temperatures, leading to a non-equilibrium migration down the column. Conversely, retention of the non-imprinted enantiomer was controlled by a thermodynamic equilibrium over the entire temperature range of the study. Variation of the structure of the analyte indicated a single leading interaction between both enantiomers of dansyl-phenylalanine and the polymer phase; a hydrogen bonding interaction between the carboxylic acid group of dansyl-phenylalanine and pyridinyl sites on the polymer. Secondary processes contributing to enantioselectivity were also deduced; a hydrogen bonding interaction occurring between the imprinted enantiomer and carboxyl sites on the polymer and a precise steric fit of the amino acid side chain into th...

Two methods for the measurement of the characteristic dissociation pressures of a system containing water vapor and two different crystalline hydrates of the pharmaceutical compound MK-0677 are described. One method involves the spectroscopic determination of water in gases equilibrated with the solids at controlled temperatures, using an infrared spectrometer. The second method utilized the extrapolated onset temperature of the transition from one hydrate to the other at controlled humidities, as observed by differential scanning calorimetry. The methods give similar results for the system of interest.

A series of amphiphilic, biodegradable polypeptide copolymers were prepared for the delivery of siRNA (short interfering ribonucleic acid). The molecular weight (or polymer chain length) of the linear polymer was controlled by reaction stoichiometry for the 11.5, 17.2, and 24.6 kDa polypeptides, and the highest molecular weight polypeptide was prepared using a sequential addition method to obtain a polypeptide having a molecular weight of 38.6 kDa. These polymers were used to prepare polymer conjugate systems designed to target and deliver an apolipoprotein B (ApoB) siRNA to hepatocyte cells and to help delineate the effect of polymer molecular weight or polymer chain length on siRNA delivery in vivo. A clear trend in increasing potency was found with increasing molecular weight of the polymers examined (at a constant polymer:siRNA (w/w) ratio), with minimal toxicity found. Furthermore, the biodegradability of these polymer conjugates was examined and demonstrates the potential of these systems as siRNA delivery vectors.

Efficient siRNA delivery is dependent not only on the ability of the delivery vehicle to target a specific organ but also on its ability to enable siRNA entry into the cytoplasm of the target cells. Polymers with endosomolytic properties are increasingly being used as siRNA delivery vehicles due to their potential to facilitate endosomal escape and intracellular delivery. Addition of disulfide bonds in the backbone of these polymers was expected to provide degradability through reduction by glutathione in cytosol. This paper describes the synthesis of new endosomolytic bioreducible poly(amido amine disulfide) polymers whose lytic potential can be masked at physiological pH, but can be restored at acidic endosomal pH. These polymer conjugates gave good in vitro knockdown (KD) and did not demonstrate cytotoxicity in a MTS assay. Efficient mRNA KD for apolipoprotein B in mouse liver was observed with these polyconjugates following intravenous dosing.

Cellulose and cellulose derivatives are biopolymers which are often used as stationary phases for the separation of enantiomers. Describing the mechanism of such separations is a difficult task due to the complexity of these phases. In the present study, we attempt to elucidate the types of interactions occurring between a diol intermediate for a LTD(4) antagonist and a tris(4-methylbenzoate)-derivatized cellulose stationary phase. Thermodynamic studies indicate that, at low temperatures, the enantioselectivity is entropy driven. At higher temperatures, the separation is enthalpy driven. DSC and IR experiments reveal that the transitions between the enthalpic and the entropic regions of the van't Hoff plots are a result of a change in conformation of the stationary phase. Investigation of chromatographic kinetic parameters reveals that, at low temperature, the second eluted enantiomer undergoes sluggish inclusion interactions. Subtle changes in the structure of the analyte indicates that π-π interactions do not contribute to enantioselectivity. Finally, molecular modeling of (R)- and (S)-diol and the stationary phase suggests that hydrogen bonding is a primary factor in the separation, and the calculated energy values obtained from the molecular modeling correlate well with the chromatographic elution order.

Potentiometric titration of (4R,5S,6S,8R,2‘S,4‘S)-3-[[2-[[(3-carboxyphenyl)amino]carbonyl]pyrrolidin-4-yl]thio]-4-methyl-6-(1-hydroxyethyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-en-2-carboxylic acid, a broad-spectrum antibiotic, in several solvent systems was used to generate values for the three dissociation constants of the compound. Simultaneous solution for the constants was employed. The proportions of the various species in solution were calculated from the dissociation constants; differences were observed between the optimum pH for isolation of the monoanion salt in aqueous−organic systems and the value calculated from aqueous titration results. Changes in the organic content of the solvent influenced the three dissociation constants to different extents.

The enthalpies of reduction of aldehydes and ketones to the corresponding alcohols have been determined by measuring the enthalpies of reaction of the carbonyl compounds and of the alcohols with lithium triethylborohydride in triglyme. The enthalpy of reduction of ethyl acetate to ethanol was determined in the same fashion. Enthalpies of formation of the carbonyl compounds were derived from the reduction data. The trends in the ΔH f values for a series of aldehydes and of ketones were examined, and the ΔH f values also were compared with the predictions derived with the MM3 molecular mechanics procedure

We report formation and characterization of the first pharmaceutically acceptable and stable molecular complex of a mono-HCl salt of Compound 1 with HCl. The novelty of this discovery is due to the fact that there is only one major basic site in the molecule. Thus this complex is reminiscent of other noncovalent crystalline forms including solvates, hydrates, cocrystals and others. To the best of our knowledge, the observed bis-HCl salt appears to be the first example of an active pharmaceutical ingredient in a form of a stable HCl complex. The paucity of stable complexes of APIs with HCl is likely due to the fact that HCl is a gas at ambient conditions and can easily evaporate compromising physical (and chemical) stability of a drug. The bis-HCl salt was chemically/physically stable at low humidity and the molecular HCl stays in the lattice until heated above 140 degrees C under nitrogen flow. Structure solution from powder diffraction using the Monte Carlo simulated annealing method as well as variable temperature ATR-FTIR suggest the possibility of weak hydrogen bonding between the molecular HCl and the nitrogen atom of the amide group. Two years later after the search for a suitable pharmaceutical salt began, the elusive conventional mono-HCl salt was obtained serendipitously concluding the lengthy quest for a regular salt. This work emphasizes the necessity to be open-minded during the salt selection process. It also highlights the difficult, lengthy and often serendipitous path of finding the most appropriate form of an API for pharmaceutical development.

The separation of [2R-[2alpha(R*),3alpha]]-5-[[2-[1-[3,5-bis-(trifluoromethyl)phenyl]ethoxy]-3(S)-4-fluorophenyl)4-morpholinyl]-methyl]-N,N-dimethyl-1H-1,2,3-triazole-4-methanamine hydrochloride from its enantiomer was achieved on an amylose tris-3,5-dimethylphenyl carbamate stationary phase. The retention of the enantiomers is dominated by weak hydrogen bonds while the enantioselectivity is governed by other kinds of interactions, e.g., inclusion in the amylose carbamate chains. Van't Hoffplots of 1nalpha vs. reciprocal temperature were non-linear and could be divided into two linear regions. One region at low temperature (5 degrees C- approximately 20 degrees C) and another one between 25 degrees C-70 degrees C with the change in slope occurring between 16 degrees C and 20 degrees C. DSC experiments suggested that the behavior can be attributed to breakage of H-bonds triggering a conformational change. Molecular simulation indicated a correlation between the interaction energies and the elution order obtained experimentally. The most retained enantiomer (R,R,S-enantiomer) interacts with the stationary phase through a hydrogen bond between the triazole proton and the C=O groups of the stationary phase, as well as through an inclusion in the cleft of the stationary phase. The other enantiomer exhibits a bifurcated H-bond between the triazolic proton and the C=O groups of the stationary phase leading to a less stable complex.

In this study, we report the use of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FT-IR) for the identification and quantitation of two polymorphs of Aprepitant, a substance P antagonist for chemotherapy-induced emesis. Mixtures of the polymorph pair were prepared by weight and ATR-FT-IR spectra of the powdered samples were obtained over the wavelength range of 700-1500 cm(-1). Significant spectral differences between the two polymorphs at 1140 cm(-1) show that ATR-FT-IR can provide definitive identification of the polymorphs. To investigate the feasibility of ATR-FT-IR for quantitation of polymorphic forms of Aprepitant, a calibration plot was constructed with known mixtures of the two polymorphs by plotting the peak ratio of the second derivative of absorbance spectra against the weight percent of form II in the polymorphic mixture. Using this novel approach, 3 wt % of one crystal form could be detected in mixtures of the two polymorphs. The accuracy of ATR-FT-IR in determining polymorph purity of the drug substance was tested by comparing the results with those obtained by X-ray powder diffractometry (XRPD). Indeed, polymorphic purity results obtained by ATR-FT-IR were found to be in good agreement with the predictions made by XRPD and compared favorably with actual values in the known mixtures. The present study clearly demonstrates the potential of ATR-FT-IR as a quick, easy, and inexpensive alternative to XRPD for the determination of polymorphic identity and purity of solid drug substances. The technique is ideally suited for polymorph analysis, because it is precise, accurate, and requires minimal sample preparation.

The pharmaceutical compound (2R,3S)-2-([(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl]oxy)-3-(4-fluorophenyl)morpholine hydrochloride (denoted here as Compound X), has been found to crystallize in at least two polymorphic forms. Using only two frequencies (1009 and 1058 cm(-1)) in the infrared, a linear (R=0.998) calibration plot, consisting of the ratio of the peak absorbances plotted against polymorph concentration, was constructed. This plot allowed the quantification of binary mixtures of polymorphs ranging from3 to approximately 100 wt% Form II in Form I. Spectra were acquired in transmission mode using mineral oil (Nujol) mull sample preparation, for reasons of compatibility with wet cake and slurry samples. The transformation of the less thermodynamically stable polymorph (Form II) to the more stable form (Form I), in stirred methyl isobutyl ketone (MIBK) slurries, was monitored spectroscopically as a function of time. Performing the experiment at various temperatures allowed the energy of activation for the process to be estimated (42 kJ/mol).

The intensity of X‐rays diffracted by amorphous material in samples of the β ‐lactam antibiotic imipenem [(5 R , 6 S )‐3‐[[2‐(formimidoyl‐amino)ethyl]thio]‐6‐[( R )‐1‐hydroxyethyl]‐7‐oxo‐1‐azabicyclo[3.2.0]hept‐2‐ene‐2‐carboxylic acid] was used to compare the crystallinity of the samples. The method makes use of the intensity in the X‐ray powder diffraction (XRPD) pattern at a diffraction angle of 28° (2Θ), at which no crystalline peaks are observed. A linear relationship ( R 2 = 0.9999) exists between amorphous intensity and crystallinity for synthetic mixtures of amorphous and crystalline imipenem. Sample preparation techniques ordinarity used in XRPD experiments were utilized.