X-ray Diffraction of Water in Polyvinylpyrrolidone.

PVP is a hydrophilic polymer commonly used as an excipient in pharmaceutical formulations. Here we have performed time-resolved high-energy X-ray scattering experiments on pellets of PVP at different humidity conditions for 1-2 days. A two-phase exponential decay in water sorption is found with a peak in the differential pair distribution function at 2.85 Å, which is attributed to the average (hydrogen bonded) carbonyl oxygen-water oxygen distance. Additional scattering measurements on powders with fixed compositions ranging from 2 to 12.3 wt % H2O were modeled with Empirical Potential Structure Refinement (EPSR). The models reveal approximately linear relations between the carbonyl oxygen-water oxygen coordination number (nOC-OW) and the water oxygen-water oxygen coordination number (nOW-OW) versus water content in PVP. A stronger preference for water-water hydrogen bonding over carbonyl-water bonding is found. At all the concentrations studied the majority of water molecules were found to be randomly isolated, but a wide distribution of coordination environments of water molecules is found within the PVP polymer strands at the highest concentrations. Overall, the EPSR models indicate a continuous evolution in structure versus water content with nOW-OW=1 occurring at ∼12 wt % H2O, i.e., the composition where, on average, each water molecule is surrounded by one other water molecule.

A High Energy X-ray Diffraction Study of Amorphous Indomethacin.

Amorphous pharmaceuticals often possess a wide range of molecular conformations and bonding arrangements. The x-ray pair distribution function (PDF) method is a powerful technique for the characterization of variations in both intra-molecular and inter-molecular packing arrangements. Here, the x-ray PDF of amorphous Indomethacin is shown to be particularly sensitive to the preferred orientations of the chlorobenzyl ring found in isomers in the crystalline state. In some cases, the chlorobenzyl ring has no preferred torsional angle in the amorphous form, while in others evidence of distinct isomer orientations are observed. Amorphous samples with no preferred torsion angles of the chlorobenzyl ring are found to favor enhanced inter-molecular hydrogen bonding, and this is reflected in the intensity of the first sharp diffraction peak. These significant variations in structure rule out amorphous Indomethacin as a possible standard for x-ray PDF measurements. At high humidity, time resolved PDF's for >40 h reveal water molecules forming hydrogen bonds with Indomethacin molecules. A simple linear hydrogen bond model indicates that water molecules in the wet amorphous form have similar hydrogen bond strengths to those found between Indomethacin dimers or chains in the dry amorphous form.

Using containerless methods to develop amorphous pharmaceuticals.

BACKGROUND: Many pipeline drugs have low solubility in their crystalline state and require compounding in special dosage forms to increase bioavailability for oral administration. The use of amorphous formulations increases solubility and uptake of active pharmaceutical ingredients. These forms are rapidly gaining commercial importance for both pre-clinical and clinical use. METHODS: Synthesis of amorphous drugs was performed using an acoustic levitation containerless processing method and spray drying. The structure of the products was investigated using in-situ high energy X-ray diffraction. Selected solvents for processing drugs were investigated using acoustic levitation. The stability of amorphous samples was measured using X-ray diffraction. Samples processed using both spray drying and containerless synthesis were compared. RESULTS: We review methods for making amorphous pharmaceuticals and present data on materials made by containerless processing and spray drying. It was shown that containerless processing using acoustic levitation can be used to make phase-pure forms of drugs that are known to be difficult to amorphize. The stability and structure of the materials was investigated in the context of developing and making clinically useful formulations. CONCLUSIONS: Amorphous compounds are emerging as an important component of drug development and for the oral delivery of drugs with low solubility. Containerless techniques can be used to efficiently synthesize small quantities of pure amorphous forms that are potentially useful in pre-clinical trials and for use in the optimization of clinical products. GENERAL SIGNIFICANCE: Developing new pharmaceutical products is an essential enterprise to improve patient outcomes. The development and application of amorphous pharmaceuticals to increase absorption is rapidly gaining importance and it provides opportunities for breakthrough research on new drugs. There is an urgent need to solve problems associated with making formulations that are both stable and that provide high bioavailability. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

4-Aminopyridine derivatives enhance impulse conduction in guinea-pig spinal cord following traumatic injury.

4-Aminopyridine (4-AP), a potassium channel blocker, is capable of restoring conduction in the injured spinal cord. However, the maximal tolerated level of 4-AP in humans is 100 times lower than the optimal dose in in vitro animal studies due to its substantially negative side effects. As an initial step toward the goal of identifying alternative potassium channel blockers with a similar ability of enhancing conduction and with fewer side effects, we have synthesized structurally distinct pyridine-based blockers. Using isolated guinea-pig spinal cord white matter and a double sucrose gap recording device, we have found three pyridine derivatives, N-(4-pyridyl)-methyl carbamate (100 microM), N-(4-pyridyl)-ethyl carbamate (100 microM), and N-(4-pyridyl)-tertbutyl (10 microM) can significantly enhance conduction in spinal cord white matter following stretch. Similar to 4-AP, the derivatives did not preferentially enhance conduction based on axonal caliber. Unlike 4-AP, the derivatives did not change the overall electrical responsiveness of axons to multiple stimuli, indicating the axons recruited by the derivatives conducted in a manner similar to healthy axons. These results demonstrate the ability of novel constructs to serve as an alternative to 4-AP for the purpose of reversing conduction deficits.

Chemical reactivity in solid-state pharmaceuticals: formulation implications.

Solid-state reactions that occur in drug substances and formulations include solid-state phase transformations, dehydration/desolvation, and chemical reactions. Chemical reactivity is the focus of this chapter. Of particular interest are cases where the drug-substance may be unstable or react with excipients in the formulation. Water absorption can enhance molecular mobility of solids and lead to solid-state reactivity. Mobility can be measured using various methods including glass transition (T(g)) measurements, solid-state NMR, and X-ray crystallography. Solid-state reactions of drug substances can include oxidation, cyclization, hydrolysis, and deamidation. Oxidation studies of vitamin A, peptides (DL-Ala-DL-Met, N-formyl-Met-Leu-Phe methyl ester, and Met-enkaphalin acetate salt), and steroids (hydrocortisone and prednisolone derivatives) are discussed. Cyclization reactions of crystalline and amorphous angiotensin-converting enzyme (ACE) inhibitors (spirapril hydrochloride, quinapril hydrochloride, and moexipril) are presented which investigate mobility and chemical reactivity. Examples of drug-excipient interactions, such as transacylation, the Maillard browning reaction, and acid base reactions are discussed for a variety of compounds including aspirin, fluoxitine, and ibuprofen. Once solid-state reactions are understood in a pharmaceutical system, the necessary steps can be taken to prevent reactivity and improve the stability of drug substances and products.

Conformational color polymorphism and control of crystallization of 5-methyl-2-[(4-methyl-2-nitrophenyl)amino]-3-thiophenecarbonitrile.

5-Methyl-2-[(4-methyl-2-nitrophenyl)amino]-3-thiophenecarbonitrile is an example of conformational and color polymorphism. The compound crystallizes in red (R), dark red (DR), light red (LR), and orange (O) modifications. There are two specific goals for this study. One is to characterize the complex thermodynamic relationship among these four known forms, and the other is to use the knowledge of the thermodynamic relationship to control the crystallization of these forms. The different forms were characterized by X-ray powder diffractometry as well as Fourier-transform infrared (FT-IR) and Raman spectroscopy; their complex thermodynamic relationships were determined by thermal analysis, solubility measurements, and slurry conversion studies. According to the solubility results, all forms are enantiotropically related: R is the thermodynamically most stable form above 60 degrees C, O is the most stable form between room temperature and 60 degrees C, LR is the most stable form below -15 degrees C, and DR is metastable throughout the entire temperature range. DR, LR, and O have very similar free energy at ambient temperature, which is the reason for the complex transition behavior. Finally, a schematic energy-temperature diagram was constructed that combines all experimental data in a comprehensive thermodynamic picture and provides insights into how to control the crystallization of the individual forms.

Effects of lyophilization on the physical characteristics and chemical stability of amorphous quinapril hydrochloride.

PURPOSE: To prepare amorphous quinapril hydrochloride (QHCl) by lyophilization and to compare its physical characteristics and chemical stability as a function of the initial pH of the pre-lyophilized solution. METHODS: Amorphous QHCl samples were prepared by lyophilization from aqueous solutions. Solid-state characteristics were evaluated by DSC, PXRD, and optical microscopy. Chemical degradation was monitored by an HPLC assay. RESULTS: Amorphous QHCl samples obtained from lyophilization exhibited variable glass transition temperatures, depending on the pH and/or concentration of the starting aqueous solutions. Neutralized quinapril (Q) in the amorphous form, which has a Tg of 51 degrees C, lower than that of its HCl salt (91 degrees C), was significantly more reactive than QHCl at the same temperature. The Tg of lyophilized samples prepared at various initial pH values correlated well with values predicted for mixtures of QHCl and Q. Their different reaction rates were related to their glass transition temperature, consistent with the results from earlier studies obtained with amorphous samples made by precipitation from an organic solution and grinding of the crystal solvate. CONCLUSIONS: Lyophilization of different QHCl solutions produces mixtures of amorphous QHCl and its neutralized form Q, with Tg values intermediate to the values of QHCl and Q. As the fraction of Q increases the overall rate of chemical degradation increases relative to QHCl alone, primarily due to the increase in molecular mobility induced by the plasticizing effects of Q.

Accelerated fluid bed drying using NIR monitoring and phenomenological modeling.

A "fast-drying" method to accelerate the fluid bed drying process is presented. It relies on concepts of heat and mass transfer with real-time near-infrared (NIR) monitoring of moisture. Triplicate trials show that fast drying can reduce granulation drying time by half over single-temperature cycles. The product is equivalent in every way tested to material made using a conventional cycle even though the inlet temperature throughout the constant-rate stage was higher than the melting point of the compound. Tablets made from the fast-dried granulation exhibit equivalent physical characteristics to tablets made from granulations dried at a single, lower temperature.

Physical characteristics and chemical degradation of amorphous quinapril hydrochloride.

This study was designed to investigate the relationships between the solid-state chemical instability and physical characteristics of a model drug, quinapril hydrochloride (QHCl), in the amorphous state. Amorphous QHCl samples were prepared by rapid evaporation from dichloromethane solution and by grinding and subsequent heating of the crystalline form. Physical characteristics, including the glass transition temperature and molecular mobility, were determined using differential scanning calorimetry, thermogravimetric analysis, powder x-ray diffractometry, polarizing microscopy, scanning electron microscopy, and infrared spectroscopy. The amorphous form of QHCl, produced by both methods, has a T(g) of 91 degrees C. Isothermal degradation studies showed that cyclization of QHCl occurred at the same rate for amorphous samples prepared by the two methods. The activation energy was determined to be 30 to 35 kcal/mol. The rate of the reaction was shown to be affected by sample weight, dilution through mixing with another solid, and by altering the pressure above the sample. The temperature dependence for chemical reactivity below T(g) correlated very closely with the temperature dependence of molecular mobility. Above T(g), however, the reaction was considerably slower than predicted from molecular mobility. From an analysis of all data, it appears that agglomeration and sintering of particles caused by softening of the solid, particularly above T(g), and a resulting reduction of the particle surface/volume ratio play a major role in affecting the reaction rate by decreasing the rate of removal of the gaseous HCl product.

Effects of freeze-dry processing conditions on the crystallization of pentamidine isethionate.

The results of this study show that pentamidine isethionate (PI) can exist in at least four crystalline forms-three anhydrates designated as forms A, B, and C, and a trihydrate. Form C is the high-temperature modification, produced by heating forms A, B, and the trihydrate above 130 degrees C and cannot be produced under actual lyophilization conditions. The crystal forms of PI present after freeze-drying depend on the initial solution concentration and the thermal history of freezing. At low concentrations of PI (4% and less), form A is observed regardless of freezing method. At a higher concentration (10%), the crystal forms observed are a function of the freezing method. Three freezing methods were used to effect different cooling rates: (1) cooling on the shelf to 2 degrees C and holding for 3 h prior to decreasing the temperature to -45 degrees C, (2) directly cooling on the shelf from room temperature to -45 degrees C, and (3) dipping the vials in liquid nitrogen. The results show that form A, form B, or a mixture of both forms are present in the freeze-dried solid depending upon whether the trihydrate crystallizes during freezing or not. Since form B can only be produced by dehydration of the trihydrate at low temperature, the presence of this form in the freeze-dried powders depends on the nucleation and growth of the trihydrate during freezing. Photostability studies have demonstrated marked differences between freeze-dried solids frozen under different conditions. The results underscore the importance of recognizing that seemingly subtle differences in processing conditions can have a significant impact on critical quality attributes of freeze-dried products.

Solid-state investigations of erythromycin A dihydrate: structure, NMR spectroscopy, and hygroscopicity.

The crystal structures of the commercially available form of erythromycin A dihydrate and clarithromycin anhydrate, in addition to the structure of erythromycin B dihydrate, are reported in this paper. In light of the crystallographic data, analysis of the structural information provides insight into the physical properties of these pharmaceuticals. The propensity of these pharmaceuticals to form solvated structures is discussed and the hygroscopicity of erythromycin A dihydrate is investigated. Solid-state 13C NMR was used to monitor changes that occur when the dihydrate form of erythromycin A is stored under conditions of low relative humidity. Although erythromycin A dihydrate retains its crystallographic order at low humidity, as indicated by its X-ray powder diffraction pattern, the local chemical environment is dramatically influenced by the loss of the water molecules and results in dramatic changes in its solid-state 13C NMR spectrum.

Impermeant antitumor sulfonylurea conjugates that inhibit plasma membrane NADH oxidase and growth of HeLa cells in culture. Identification of binding proteins from sera of cancer patients.

The antitumor sulfonylurea LY237868 (N-(4-aminophenyl-sulfonyl)-N'-(4-chlorophenyl)urea) was conjugated through the A ring to alpha-cyclodextrin or agarose bead material (Affigel 10) to prepare impermeant conjugates for activity measurements and affinity isolation of binding proteins from serum. When conjugated to alpha-cyclodextrin, the resulting LY237868 conjugate inhibited both NADH oxidase activity and growth of HeLa cells in culture. The conjugate was at least one order of magnitude more potent as an inhibitor than the parent compound. These findings confirm previous results that demonstrate an antitumor sulfonylurea-binding protein with NADH oxidase activity at the external plasma membrane surface of HeLa cells that is shed into culture media conditioned by growth of HeLa cells. A comparable activity, responsive to sulfonylurea, was present in sera of cancer patients. LY237868 conjugated to agarose beads as the affinity support bound a large number of serum proteins. However, compared to serum from normal patients, the affinity support bound two proteins of M(r) approx. 33.5 and 29.5 not found in sera of normal patients. The 33.5 kDa protein from human sera reacted with antisera to a 33.5 kDa protein from culture media conditioned by growth of HeLa cells that blocked and immunoprecipitated the sulfonylurea-responsive activity from HeLa cell plasma membranes. The results point to the 33.5 kDa protein from cancer patient sera that bound to the sulfonylurea affinity support as representing the circulating equivalent of the previously identified 34 kDa sulfonylurea-binding protein, with NADH oxidase activity at the external cell surface of cultured HeLa cells and a corresponding 33.5 kDa protein shed into culture media conditioned by growth of HeLa cells.

Glycine crystallization during freezing: the effects of salt form, pH, and ionic strength.

PURPOSE: The purpose of the study is to characterize glycine crystallization during freezing of aqueous solutions as a function of the glycine salt form (i.e., neutral glycine, glycine hydrochloride, and sodium glycinate), pH, and ionic strength. METHODS: Crystallization was studied by thermal analysis, microscopy, x-ray diffraction, and pulsed Fourier transform nmr spectroscopy. RESULTS: A solution of neutral glycine with no additives undergoes rapid secondary crystallization during freezing, forming the beta polymorph, with a eutectic melting temperature of -3.4 degrees C. Glycine hydrochloride solutions undergo secondary crystallization relatively slowly, and the eutectic melting temperature is -28 degrees C. Sodium glycinate crystallizes from frozen solution at an intermediate rate, forming a eutectic mixture with a melting temperature of -17.8 degrees C. Where secondary crystallization does not occur rapidly, a complex glass transition is observed in the -70 degrees to -85 degrees C temperature range in the DSC thermograms of all systems studied. Rates of secondary crystallization and the type of crystal formed are influenced by solution pH relative the the pKs of glycine, and also by the change in ionic strength caused by adjustment of pH. Increased ionic strength significantly slows the crystallization of neutral glycine and promotes formation of the gamma polymorph. Thermal treatment or extended holding times during the freezing process may be necessary in order to promote secondary crystallization and prevent collapse during freeze drying. CONCLUSIONS: The results underscore the importance of recognizing that seemingly minor changes in formulation conditions can have profound effects on the physical chemistry of freezing and freeze drying.

Hydration and dehydration of crystalline and amorphous forms of raffinose.

The trisaccharide raffinose was prepared in its crystal pentahydrate, anhydrous methanolate, and amorphous forms and evaluated with regard to dehydration and hydration properties at various temperatures and relative humidities. The pentahydrate, when stored at relative humidities (RHs) of < 60% but > 10%, showed no loss of water after 3 months of storage at 30 degrees C. When stored below 10% RH, only one water molecule could be removed over a period of 3 months, whereas within 24 h at 30 degrees C in a vacuum oven, two water molecules were removed with no change in crystal structure. Increasing the temperature to 60 degrees C progressively removed the remaining three molecules, causing the crystal, however, to collapse into an amorphous form identical to one prepared by lyophilization. Rehydration at 30 degrees C, which was sufficient to reduce the glass transition temperature to < 30 degrees C, rapidly restored the pentahydrate crystal structure. Rehydration of the methanolate also restored the pentahydrate structure. The significant amount of water accommodated by raffinose in both the crystalline and amorphous forms would appear to make it a potentially useful water scavenger in certain types of dosage forms.

Sequence specific cleavage of messenger RNA by a modified ribonuclease H.

Ribonuclease H (RNase H) is an endonuclease that cleaves only the RNA strand of an RNA-DNA hybrid to produce 5'-phosphate and 3'-hydroxy termini and lacks useful sequence specific recognition properties. A mutant form of the E. coli enzyme has been prepared that is suited for selective chemical modification at a site proximal to the substrate binding region. The chemical derivatization involves the formation of a disulfide linkage to a modified octadeoxyribonucleotide. The conjugate retains only 0.3% of the normal sequence independent RNase H activity demonstrating that substrate recognition can be modulated by a covalent appendage. A beta-globin RNA transcript containing a sequence complementary to that of the octadeoxyribonucleotide was cleaved in a catalytic fashion to two products upon treatment with the conjugate. The selectivity in the phosphodiester bond cleavage mediated by the conjugate was found to be different than that displayed by the nonderivatized enzyme. These results demonstrate the potential of semi-synthetic RNase H conjugates for mechanistic studies and their application as RNA targeted diagnostic or therapeutic agents.

Solid-state nuclear magnetic resonance (NMR) spectra of pharmaceutical dosage forms.

Solid-state 13C NMR spectra of tablets or capsules of prednisolone, enalapril maleate, lovastatin, simvastatin, ibuprofen, flurbiprofen, mefenamic acid, indomethacin, diflunisal, sulindac, and piroxicam were obtained in the CP/MAS mode at 50 MHz. These studies show that (1) solid-state NMR spectroscopy can detect the active ingredients in low-dose tablets and capsules; (2) the use of interrupted decoupling often results in suppression of resonances due to excipients, thereby allowing better detection of resonances from the drug; and (3) the technique permits discrimination between two prednisolone polymorphs present in tablets obtained from various manufacturers even though the tablets contain only approximately 5% (w/w) of the drug.

Tubulosine: an antitumor constituent of Pogonopus speciosus.

From the antitumor-bioactive sap of Pogonopus speciosus, tubulosine [1] was isolated, by activity-directed fractionation using the brine shrimp lethality test, as the major antitumor constituent. 1H-nmr assignments, obtained from HETCOR and COSY, and X-ray crystallographic results are reported for the first time. Psychotrine [2] was also isolated, and its spectral data are also reported.